Cellulase Variants

ABSTRACT

The present invention relates to a method for improving the properties of a cellulolytic enzyme by amino acid substitution, deletion or insertion, the method comprising the steps of:
     a. constructing a multiple alignment of at least two amino acid sequences known to have three-dimensional structures similar to endoglucanase V (EGV) from  Humicola insolens  known from Protein Data Bank entry 4ENG;   b. constructing a homology-built three-dimensional structure of the cellulolytic enzyme based on the structure of the EGV;   c. identifying amino acid residue positions present in a distance from the substrate binding cleft of not more than 5 Å;   d. identifying surface-exposed amino acid residues of the enzyme;   e. identifying all charged or potentially charged amino acid residue positions of the enzyme;   f. choosing one or more positions wherein the amino acid residue is to be substituted, deleted or where an insertion is to be provided; and   g. carrying out the substitution, deletion or insertion by using conventional protein engineering techniques. Also described are cellulase variants obtained by this method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application no. 10/919,195filed Aug. 16, 2004, which is a continuation of U.S. application no.09/261,329 filed Mar. 3, 1999, which is a continuation of PCT/DK97/00393filed Sep. 17, 1997, which claims priority under 35 U.S.C. 119 of Danishapplication 1013/96 filed Sep. 17, 1996, the contents of which are fullyincorporated herein by reference.

SEQUENCE LISTING

The present application contains information in the form of a sequencelisting, which is appended to the application and also submitted on adata carrier accompanying this application. The data carrier is fullyincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to cellulase variants, i.e.,endo-beta-1,4-glucanase variants, derived from a parental cellulase,i.e., endo-beta-1,4-glucanase, by substitution, insertion and/ordeletion, which variant has a catalytic core domain, in which thevariant at position 5 holds an alanine residue (A), a serine residue(S), or a threonine residue (T); at position 8 holds a phenylalanineresidue (F), or a tyrosine residue (Y), at position 9 holds aphenylalanine residue (F), a tryptophan residue (W), or a tyrosineresidue (Y); at position 10 holds an aspartic acid residue (D), and atposition 121 holds an aspartic acid residue (D).

2. Description of Related Art

Cellulases or cellulolytic enzymes are enzymes involved in hydrolysis ofcellulose. In the hydrolysis of native cellulose, it is known that thereare three major types of cellulase enzymes involved, namelycellobiohydrolase (1,4-beta-D-glucan cellobiohydrolase, EC 3.2.1.91),endo-beta-1,4-glucanase (endo-1,4-beta-D-glucan 4-glucanohydrolase, EC3.2.1.4) and beta-glucosidase (EC 3.2.1.21).

Especially the endo-beta-1,4-glucanases (EC No. 3.2.1.4) constitute aninteresting group of hydrolases for the mentioned industrial uses.Endoglucanases catalyses endo hydrolysis of 1,4-beta-D-glycosidiclinkages in cellulose, cellulose derivatives (such as carboxy methylcellulose and hydroxy ethyl cellulose), lichenin, beta-1,4 bonds inmixed beta-1,3 glucans such as cereal beta-D-glucans or xyloglucans andother plant material containing cellulosic parts. The authorized name isendo-1,4-beta-D-glucan 4-glucano hydrolase, but the abbreviated termendoglucanase is used in the present specification. Reference can bemade to T.-M. Enveri, “Microbial Cellulases” in W. M. Fogarty, MicrobialEnzymes and Biotechnology, Applied Science Publishers, p. 183-224(1983); Methods in Enzymology, 1988, Vol. 160, pp. 200-391 (edited byWood, W. A. and Kellogg, S. T.); Béguin, P., “Molecular Biology ofCellulose Degradation”, Annu. Rev. Microbiol., 1990, Vol. 44, pp.219-248; Béguin, P. and Aubert, J-P., “The biological degradation ofcellulose”, FEMS Microbiology Reviews, 1994, Vol. 13, pp. 25-58;Henrissat, B., “Cellulases and their interaction with cellulose”,Cellulose, 1994, Vol. 1, pp. 169-196.

Cellulases are synthesized by a large number of microorganisms whichinclude fungi, actinomycetes, myxobacteria and true bacteria but also byplants. Especially endoglucanases of a wide variety of specificitieshave been identified.

A very important industrial use of cellulolytic enzymes is the use fortreatment of cellulosic textile or fabric, e.g., as ingredients indetergent compositions or fabric softener compositions, forbio-polishing of new fabric (garment finishing), and for obtaining a“stone washed” look of cellulose-containing fabric, especially denim,and several methods for such treatment have been suggested, e.g., inGB-A-1 368 599, EP-A-0 307 564 and EP-A-0 435 876, WO 91/17243, WO91/10732, WO 91/17244, PCT/DK95/000108 and PCT/DK95/00132. Anotherimportant industrial use of cellulolytic enzymes is the use fortreatment of paper pulp, e.g., for improving the drainage or fordeinking of recycled paper.

It is also known that cellulases may or may not have a cellulose bindingdomain (a CBD). The CBD enhances the binding of the enzyme to acellulose-containing fiber and increases the efficacy of the catalyticactive part of the enzyme

Fungi and bacteria produces a spectrum of cellulolytic enzymes(cellulases) which, on the basis of sequence similarities (hydrophobiccluster analysis), can be classified into different families of glycosylhydrolases [Henrissat B & Bairoch A; Biochem. J., 1993, 293; 781-788].At present are known cellulases belonging to the families 5, 6, 7, 8, 9,10, 12, 26, 44, 45, 48, 60, and 61 of glycosyl hydrolases.

Industrially well-performing endo-beta-1,4-glucanases are described in,e.g., WO 91/17243, WO 91/17244 and WO 91/10732, and specific cellulasevariants are described in WO 94/07998.

It is an object of the present invention to provide novel variants ofcellulolytic enzymes, which variants, when compared to the parentalenzyme, show improved performance.

SUMMARY OF THE INVENTION

In a cellulolytic enzyme useful in industrial processes, i.e., anendo-1,4-glucanase, a number of amino acid residue positions importantfor the properties of the enzyme and thereby for the performance thereofin these processes has been identified.

Accordingly, in a first aspect the present invention provides a methodfor improving the properties of a cellulolytic enzyme by amino acidsubstitution, deletion or insertion, the method comprising the steps of:

a. constructing a multiple alignment of at least two amino acidsequences known to have three-dimensional structures similar toendoglucanase V (EGV) from Humicola insolens known from Protein DataBank entry 4ENG;

b. constructing a homology-built three-dimensional structure of thecellulolytic enzyme based on the structure of the EGV;

c. identifying amino acid residue positions present in a distance fromthe substrate binding cleft of not more than 5 Å;

d. identifying surface-exposed amino acid residues of the enzyme;

e. identifying all charged or potentially charged amino acid residuepositions of the enzyme;

f. choosing one or more positions wherein the amino acid residue is tobe substituted, deleted or where an insertion is to be provided;

g. carrying out the substitution, deletion or insertion by usingconventional protein engineering techniques.

By using the method of the invention, it is now possible effectively totransfer desirable properties from one cellulase to another by proteinengineering methods which are known per se.

More particular the invention provides cellulase variants improved withrespect to altered (increased or decreased) catalytic activity; and/oraltered sensitivity to anionic tensides; and/or altered pH optimum andpH profile activity-wise as well as stability-wise.

Accordingly, in a further aspect, the invention provides a cellulasevariant derived from a parental cellulase by substitution, insertionand/or deletion, which variant has a catalytic core domain, in which thevariant

at position 5 holds an alanine residue (A), a serine residue (S), or athreonine residue (T);

at position 8 holds a phenylalanine residue (F), or a tyrosine residue(Y);

at position 9 holds a phenylalanine residue (F), a tryptophan residue(W), or a tyrosine residue (Y);

at position 10 holds an aspartic acid residue (D); and

at position 121 holds an aspartic acid residue (D) (cellulasenumbering).

DETAILED DISCLOSURE OF THE INVENTION Cellulase Variants

The present invention provides new cellulase variants derived from aparental cellulase by substitution, insertion and/or deletion. Acellulase variant of this invention is a cellulase variant or mutatedcellulase, having an amino acid sequence not found in nature. Thecellulase variants of the invention show improved performance, inparticular with respect to increased catalytic activity; and/or alteredsensitivity to anionic tensides; and/or altered pH optimum; and/oraltered thermostability.

Formally the cellulase variant or mutated cellulase of this inventionmay be regarded a functional derivative of a parental cellulase (i.e.,the native or wild-type enzyme), and may be obtained by alteration of aDNA nucleotide sequence of the parental gene or its derivatives,encoding the parental enzyme. The cellulase variant or mutated cellulasemay be expressed and produced when the DNA nucleotide sequence encodingthe cellulase variant is inserted into a suitable vector in a suitablehost organism. The host organism is not necessarily identical to theorganism from which the parental gene originated.

In the literature, enzyme variants have also been referred to as mutantsor muteins.

Amino Acids

In the context of this invention the following symbols and abbreviationsfor amino acids and amino acid residues are used:

A = Ala = Alanine C = Cys = Cysteine D = Asp = Aspartic acid E = Glu =Glutamic acid F = Phe = Phenylalanine G = Gly = Glycine H = His =Histidine I = Ile = Isoleucine K = Lys = Lysine L = Leu = Leucine M =Met = Methionine N = Asn = Asparagine P = Pro = Proline Q = Gln =Glutamine R = Arg = Arginine S = Ser = Serine T = Thr = Threonine V =Val = Valine W = Trp = Tryptophan Y = Tyr = Tyrosine B = Asx = Asp orAsn Z = Glx = Glu or Gln X = Xaa = Any amino acid * = Deletion or absentamino acid

Cellulase Numbering

In the context of this invention a specific numbering of amino acidresidue positions in cellulolytic enzymes is employed. By aligning theamino acid sequences of known cellulases, as in Table 1 below, it ispossible to unambiguously allot an amino acid position number to anyamino acid residue in any cellulolytic enzyme, if its amino acidsequence is known.

In Table 1, below, 11 selected amino acid sequences of cellulases ofdifferent microbial origin are aligned. These are (a) Humicola insolens;(b) Acremonium sp.; (c) Volutella collectotrichoides; (d) Sordariafimicola; (e) Thielavia terrestris; (f) Fusarium oxysporum; (g)Myceliophthora thermophila; (h) Crinipellis scabella; (i) Macrophominaphaseolina; (j) Pseudomonas fluorescens; (k) Ustilago maydis. Thecellulases (a-i) are described in WO 96/29397, (j) is described inGeneBank under the accession number G45498, and (k) is described inGeneBank under the accession number S81598 and in Biol. Chem.Hoppe-Seyler, 1995, 376 (10): 617-625.

Using the numbering system originating from the amino acid sequence ofthe cellulase (endo-beta-1,4-glucanase) obtained from the strain ofHumicola insolens DSM 1800, disclosed in, e.g., WO 91/17243, whichsequence is shown in the first column of Table 1, aligned with the aminoacid sequence of a number of other cellulases, it is possible toindicate the position of an amino acid residue in a cellulolytic enzymeunambiguously.

In describing the various cellulase variants produced or contemplatedaccording to the invention, the following nomenclatures are adapted forease of reference:

-   -   [Original amino acid; Position; Substituted amino acid]

Accordingly, the substitution of glutamine with histidine in position119 is designated as Q119H.

Amino acid residues which represent insertions in relation to the aminoacid sequence of the cellulase from Humicola insolens, are numbered bythe addition of letters in alphabetical order to the preceding cellulasenumber, such as, e.g., position *21aV for the “inserted” valine (V),where no amino acid residue is present, between lysine at position 21and alanine at position 22 of the amino acid sequence of the cellulasefrom Humicola insolens, cf. Table 1.

Deletion of a proline (P) at position 49 in the amino acid sequence ofthe cellulase from Humicola insolens is indicated as P49*.

Multiple mutations are separated by slash marks (“/”), e.g.,Q119H/Q146R, representing mutations in positions 119 and 146substituting glutamine (Q) with histidine (H), and glutamine (Q) acidwith arginine (R), respectively.

If a substitution is made by mutation in, e.g., a cellulase derived froma strain of Humicola insolens, the product is designated, e.g.,“Humicola insolens/*49P”.

All positions referred to in this application by cellulase numberingrefer to the cellulase numbers described above, and are determinedrelative to the amino acid sequence of the cellulase derived fromHumicola insolens, cf. Table 1, (a).

TABLE 1 Amino Acid Sequence Alignment. a b c d e f g h i j k   1 A G G GG G G T T C *   2 D S T S S S I A S N *   3 G G G G G G G G G G G   4 RH R K Q H Q V V Y M   5 S T T S S S T T T A A   6 T T T T T T T T T T T  7 R R R R R R R R R R R   8 Y Y Y Y Y Y Y Y Y Y Y   9 W W W W W W W WW W W  10 D D D D D D D D D D D  11 C C C C C C C C C C C  12 C C C C CC C C C C C  13 K K K K K K K K K K L  14 P P P P P P P P P P A  15 S SS S S S S S S H S  16 C C C C C C C C C C A  17 G A G A A S A G A G S 18 W W W W W W W W W W W  19 A D D S P S P S T S E  20 K E E G G G G GG A G  21 K K K K K K K K K N K   21a * * * * * * * * * V *  22 A A A AA A G A A P A  23 P A S S A A P S S S P  24 V V V V V V * V V L V  25 NS S N S N S S S V Y  26 Q R Q R Q A S A K S A  27 P P P P P P P P P P P 28 V V V V V A V V V L V  29 F T K L Y L Q R G Q D  30 S T T A A T A TT S A  31 C C C C C C C C C C C  32 N D D D D D D D D S K  33 A R R A AK K R I A A  34 N N N N N N N N N N D  35 F N N N F D D G D N G  36 Q SN N Q N N N N T V  37 R P P P R P P T A R T  38 I L L L L I F L Q L L 39 T S A N S S N G T S I  40 D P S D D N D P P D D  41 F * * A F T G *S V S  42 D G T N N N G * D S K   42a * * * * * * S D L * K  43 A A A VV A T V L V D  44 K V R K Q V R K K G P  45 S S S S S N S S S S S  46 GG G G G G G G S S G  47 C C C C C C C C C C Q  48 E D D D N E D D D D S 49 P P S * * G A S * * G   49a * * * * * * * * * * C  49b * * * * * * * * * * N  50 G N N G G G G G G G G  51 G G G G G G GG G G G  52 V V V S S S S T S G N  53 A A A A A A A S A G K  54 Y F Y YY Y Y F Y Y F  55 S T T T S A M T Y M M  56 C C C C C C C C C C C  57 AN N A A T S A S W S  58 D D D N D N S N N D C  59 Q N N N Q Y Q N Q K M 60 T Q Q S T S S G G I Q  61 P P P P P P P P P P P  62 W W W W W W W FW F F  63 A A A A A A A A A A D  64 V V V V V V V I V V D  65 N N N N NN S D N S E  66 D N D D D D D N D P T  67 D N N N N E E N S T D  68 F VL L L L L T L L P  69 A A A A A A S A S A T  70 L Y Y Y Y Y Y Y Y Y L 71 G G G G G G G G G G A  72 F F F F F F W F F Y F  73 A A A A A A A AA A G  74 A A A A A A A A A A F  75 T T T T T T V A A T G  76 S A A K SK K H K S A  77 I F F L I I L L L S F  78 A P S S A S A A S G T  79 G GG G G G G G G D T  80 S G G G G G S S K V G  81 N N S T S S S S Q * Q 82 E E E E E E E E E * E  83 A A A S S A S A T * S  84 G S S S S S Q AD * D  85 W W W W W W W W W * T  86 C C C C C C C C C C D  87 C C C C CC C C C G C  88 A A A A A A A Q G R A  89 C C C C C C C C C C C  90 Y YY Y Y Y Y Y Y Y F  91 E A A A A A E E K Q Y  92 L L L L L L L L L L A 93 T Q Q T T T T T T Q E  94 F F F F F F F F F F F  95 T T T T T T T TT T E   95a * * * * * * * * * G *   95b * * * * * * * * * S *  95c * * * * * * * * * S *   95d * * * * * * * * * Y *  95e * * * * * * * * * N *   95f * * * * * * * * * A *  95g * * * * * * * * * P *   95h * * * * * * * * * G H  95i * * * * * * * * * D D   95j * * * * * * * * * P A  95k * * * * * * * * * G Q  96 S S S S S T S S S S G  97 G G G G G G GG T A K  98 P P P P P P P P A A A  99 V V V V V V V V V L M 100 A A A SA K A V S A K 101 G G G G G G G G G G R 102 K K K K K K K K K K N 103 KT T T T K K K Q T K 104 M M M L M M M L M M L 105 V V V V V I I T I I I106 V V V V V V V V V V F 107 Q Q Q Q Q Q Q Q Q Q Q 108 S S S S S S A VI A V 109 T T T T T T T T T T T 110 S N N S S N N N N N N 111 T T T T TT T T T I V 112 G G G G G G G G G G G 113 G G G G G G G G G Y G 114 D DD D D D D D D D D 115 L L L L L L L L L V V 116 G S S G G G G G G S Q117 S G G S S D D N N G S 118 N T N N N N N N N G Q 119 H H H H Q H H HH Q N 120 F F F F F F F F F F F 121 D D D D D D D D D D D 122 L I I L IL L L I I F 123 N Q L N A M A M A L Q 124 I M M M M M I I M V I 125 P PP P P P P P P P P 126 G G G G G G G G G G G 127 G G G G G G G G G G G128 G G G G G G G G G G G 129 V L L V V V V V V V L 130 G G G G G G G GG G G 131 I I I L I I I L I A A 132 F F F F F F F F F F F 132a * * * * * * * T * * P 133 D D D D N D N Q N N K 134 G G G G G G AG G A G 135 C C C C C C C C C C C 136 T T T K S T T P S S P 137 P P P RS S D A K A A 138 Q Q Q E Q E Q Q Q Q Q 139 F F W F F F Y F W W W 140 GG G G G G G G N G G  140a * F V * * K A S G V V 141 G T S G G A P W I SE 142 L F F L L L P N * N A 143 P P P P P G N G * A S  143a * * * * * *G * N E L  143b * * * * * * W * L L W 144 G G G G G G G G G G G 145 Q NN A A A D A N A D 146 R R R Q Q Q R Q Q Q Q 147 Y Y Y Y Y Y Y Y Y Y Y148 G G G G G G G G G G G 149 G G G G G G G G G G G 150 I T T I I I I VF F V  150a * * * * * * * * * L *  150b * * * * * * * * * A * 150c * * * * * * * * * A *  150d * * * * * * * * * C * 150e * * * * * * * * * K *  150f * * * * * * * * * Q * 150g * * * * * * * * * Q *  150h * * * * * * * * * L * 150i * * * * * * * * * G *  150j * * * * * * * * * Y * 150k * * * * * * * * * N * 151 S T T S S S H S T A K 152 S S S S S S SS D S S 153 R R R R R R K R R L A 154 N S S S D S E D S S T 155 E Q Q EQ E E Q Q Q E 156 C C C C C C C C C Y C 157 D A S D D D E S A K S 158 RE Q S S S S Q T S K 159 F L I F F Y F L L C L 160 P P P P P P P P P V P 160a * * * * * * * * * L *  160b * * * * * * * * * N * 160c * * * * * * * * * R *  160d * * * * * * * * * C * 160e * * * * * * * * * D *  160f * * * * * * * * * S * 160g * * * * * * * * * V *  160h * * * * * * * * * F * 160i * * * * * * * * * G *  160j * * * * * * * * * S * 160k * * * * * * * * * R *  160l * * * * * * * * * G * 160m * * * * * * * * * L * 161 D S S A A E E A S T K 162 A V A A P L AA K Q P 163 L L L L L L L V W L L 164 K R Q K K K K Q Q Q Q 165 P D P PP D P A A Q E 166 G G G G G G G G S G G 167 C C C C C C C C C C C 168 YH N Q Q H N Q N T K 169 W W W W W W W F W W W 170 R R R R R R R R R F R171 F Y Y F F F F F F A F 172 D D D D D D D D D E S 173 W W W W W W W WW W E 174 F F F F F F F M F F W 175 K N N K Q E Q G E E G 176 N D D N NN N G N A D 177 A A A A A A A A A A N 178 D D D D D D D D D D P 179 N NN N N N N N N N V 180 P P P P P P P P P P L 181 S N D E T D S N T S K182 F V V F F F V V V L G 183 S N S T T T T T D K S 184 F W W F F F F FW Y P 185 R R R K Q E Q R E K K 186 Q R R Q Q Q E P P E R 187 V V V V VV V V V V V 188 Q R Q Q Q Q A T T P K 189 C C C C C C C C C C C 190 P PP P P P P P P P P 191 A A A S A K S A Q A K 192 E A A E E A E Q E E S193 L L L L I L L L L L L 194 V T T T V L T T V T I 195 A N D S A D S NA T D 196 R R R R R I K I R R R 197 T S T T S S S S T S S 198 G G G G GG G G G G G 199 C C C C C C C C C M C 200 R V R K K K S V S N Q 201 R RR R R R R R R R R *Amino acid residue absent in this position CellulaseNumbering of Selected Cellulases of Different Microbial Origin (a)Humicola insolens (SEQ ID NO: 1); (b) Acremonium sp. (SEQ ID NO: 2); (c)Volutella collectotrichoides (SEQ ID NO: 3); (d) Sordaria fimicola (SEQID NO: 4); (e) Thielavia terrestris (SEQ ID NO: 5); (f) Fusariumoxysporium (SEQ ID NO: 6); (g) Myceliophthora thermophila (SEQ ID NO:7); (h) Crinipellis scabella (SEQ ID NO: 8); (i) Microphomina phaseolina(SEQ ID NO: 9); (j) Pseudomonas fluorescens (SEQ ID NO: 10); (k)Ustilago maydis (SEQ ID NO: 11).The Enzyme (endo-beta-1,4-glucanase) Variants of the Invention

The present invention relates to cellulase variants. More specificallythe present invention provides cellulase variant derived from a parentalcellulase by substitution, insertion and/or deletion, which variant hasa catalytic core domain, in which the variant

-   -   at position 5 holds an alanine residue (A), a serine residue        (S), or a threonine residue (T);    -   at position 8 holds a phenylalanine residue (F) or a tyrosine        residue (Y);    -   at position 9 holds a phenylalanine residue (F), a tryptophan        residue (W), or a tyrosine residue (Y);    -   at position 10 holds an aspartic acid residue (D); and    -   at position 121 holds an aspartic acid residue (D) (cellulase        numbering).

The endoglucanase of the invention may comprise a cellulose bindingdomain (CBD) existing as an integral part of the enzyme, or a CBD fromanother origin may be introduced into the endoglucanase thus creating anenzyme hybride. In this context, the term “cellulose-binding domain” isintended to be understood as defined by Peter Tomme et al.“Cellulose-Binding Domains: Classification and Properties” in “EnzymaticDegradation of Insoluble Carbohydrates”, John N. Saddler and Michael H.Penner (Eds.), ACS Symposium Series, No. 618, 1996. This definitionclassifies more than 120 cellulose-binding domains into 10 families(I-X), and demonstrates that CBDs are found in various enzymes such ascellulases, xylanases, mannanases, arabinofuranosidases, acetylesterases and chitinases. CBDs have also been found in algae, e.g., . .. the red alga Porphyra purpurea as a non-hydrolyticpolysaccharide-binding protein, see Tomme et al., op. cit. However, mostof the CBDs are from cellulases and xylanases, CBDs are found at the Nand C termini of proteins or are internal. Enzyme hybrids are known inthe art, see, e.g., WO 90/00609 and WO 95/16782, and may be prepared bytransforming into a host cell a DNA construct comprising at least afragment of DNA encoding the cellulose-binding domain ligated, with orwithout a linker, to a DNA sequence encoding the endoglucanase andgrowing the host cell to express the fused gene.

Enzyme hybrids may be described by the following formula:

CBD-MR-X or X-MR-CBD

wherein CBD is the N-terminal or the C-terminal region of an amino acidsequence corresponding to at least the cellulose-binding domain, MR isthe middle region (the linker), and may be a bond, or a short linkinggroup preferably of from about 2 to about 100 carbon atoms, morepreferably of from 2 to 40 carbon atoms; or is preferably from about 2to about 100 amino acids, more preferably of from 2 to 40 amino acids;and X is an N-terminal or C-terminal region of the enzyme according tothe invention.

The Method of the Invention

In another aspect, the present invention relates to a method forimproving the properties of a cellulolytic enzyme by amino acidsubstitution, deletion or insertion, the method comprising the steps of:

a. constructing a multiple alignment of at least two amino acidsequences known to have three-dimensional structures similar toendoglucanase V (EGV) from Humicola insolens known from Protein DataBank entry 4ENG;

b. constructing a homology-built three-dimensional structure of thecellulolytic enzyme based on the structure of the EGV;

c. identifying amino acid residue positions present in a distance fromthe substrate binding cleft of not more than 5 Å;

d. identifying surface-exposed amino acid residues of the enzyme;

e. identifying all charged or potentially charged amino acid residuepositions of the enzyme;

f. choosing one or more positions wherein the amino acid residue is tobe substituted, deleted or where an insertion is to be provided;

g. carrying out the substitution, deletion or insertion by usingconventional protein engineering techniques.

Step f. of the method is preferably carried out by choosing positionswhich, as a result of the alignment of step a., carry the same aminoacid residue in a majority of the aligned sequences; more preferably inat least 63% of the aligned sequences; even more preferably positionswhich, in the aligned sequences, carries different amino acid residues,cf. below.

In a preferred embodiment, the specific activity of the cellulase can beimproved, preferably by carrying out a substitution, deletion orinsertion at amino acid residue positions present in a distance from thesubstrate binding cleft of not more than 5 Å, more preferably not morethan 3 Å, even more preferably not more than 2 Å. It is believed thatresidues present in a distance of not more than 2.5 Å are capable ofbeing in direct contact with the substrate.

In another preferred embodiment, the pH activity profile, the pHactivity optimum, the pH stability profile, or the pH stability optimumof the cellulase can be altered, preferably by carrying out asubstitution, deletion or insertion at amino acid residue positionspresent either in a distance from the substrate binding cleft of notmore than 5 Å, more preferably not more than 3 Å, even more preferablynot more than 2.5 Å; or at surface-exposed amino acid residue positionsof the enzyme, thereby altering the electrostatic environment eitherlocally or globally. It is preferred to perform a substitution involvinga charged or potentially charged residue, this residue either being theoriginal residue or the replacement residue. In the present context,charged or potentially charged residues are meant to include: Arg, Lys,His, Cys (if not part of a disulfide bridge), Tyr, Glu, and Asp.

In yet another preferred embodiment, the stability of the cellulase inthe presence of an anionic tenside or anionic detergent component can bealtered, preferably by carrying out a substitution, deletion orinsertion at surface-exposed amino acid residue positions of the enzyme,thereby altering the electrostatic environment either locally orglobally. It is preferred to perform a substitution involving a chargedor potentially charged residue, this residue either being the originalresidue or the replacement residue. In the present context, charged orpotentially charged residues are meant to include: Arg, Lys, His, Cys(if not part of a disulfide bridge), Tyr, Glu, and Asp. Mutationstowards a more negatively charged amino acid residue result in improvedstability of the cellulase in the presence of an anionic tenside,whereas mutations towards a more positively charged aa residue decreasesthe stability of the cellulase towards anionic tensides.

Further, cellulase variants comprising any combination of two or more ofthe amino acid substitutions, deletions or insertions disclosed hereinare also within the scope of the present invention, cf. the exemplifiedvariants.

Multiple Sequence Alignment of Cellulases

The multiple sequence alignment is performed using the Pileup algorithmas implemented in the Wisconsin Sequence Analysis Package version8.1-UNIX (GCG, Genetics Computer Group, Inc.). The method used issimilar to the method described by Higgens and Sharp (CARBIOS, 1989, 5,151-153). A gap creation penalty of 3.0 and a gap extension penalty of0.1 are used together with a scoring matrix as described in Nucl. AcidsRes. 1986, 14 (16), 6745-6763 (Dayhoff table (Schwartz, R. M. andDayhoff, M. O.; Atlas of Protein Sequence and Structure (Dayhoff, M. O.Ed.); National Biomedical Research Foundation, Washington D.C., 1979,353-358) rescaled by dividing each value by the sum of its row andcolumn, and normalizing to a mean of 0 and standard deviation of 1.0.The value for FY (Phe-Tyr)=RW=1.425. Perfect matches are set to 1.5 andno matches on any row are better than perfect matches).

Pair-Wise Sequence Alignment of Cellulases

A pair-wise sequence alignment is performed using the algorithmdescribed by Needleman & Wunsch (J. Mol. Biol., 1970, 48, 443-453), asimplemented in the GAP routine in the Wisconsin Sequence AnalysisPackage (GCG). The parameters used for the GAP routine are the same asmentioned for the Pileup routine earlier,

Pair-Wise Sequence Alignment of Cellulases with Forced Pairing

A pair-wise sequence alignment with forced pairing of residues isperformed using the algorithm described by Needleman & Wunsch (J. Mol.Biol., 1970, 48, 443-453), as implemented in the GAP routine in theWisconsin Sequence Analysis Package (GCG). The parameters used for theGAP routine are the same as mentioned for the Pileup routine earlier,where the scoring matrix is modified to incorporate a residue named Xwhich symbolizes the residues to be paired. The diagonal value for Xpaired with X is set to 9.0 and all off diagonal values involving X isset to 0.

Complex between Humicola insolens Endoglucanase and Celloheptaose

Based on the X-ray structure of the core domain of the Humicola insolensEGV endoglucanase inactive variant (D10N) in complex with cellohexaose(Davies et. al.; Biochemistry, 1995, 34, 16210-12220, PDB entry 4ENG) amodel of the structure of the native Humicola insolens EGV endoglucanasecore domain in complex with celloheptaose is build using the followingsteps:

1. Using the Biopolymer module of the Insight II 95.0 (Insight II 95.0User Guide, October 1995, San Diego: Biosym/MSI, 1995) replace N10 witha aspartic acid.2. Make a copy of the sugar unit occupying subsite −3 by copying all themolecule and delete the extra atoms. Manually move the new sugar unit tobest fit the unoccupied −1 binding site, Create the bonds to bind thenew sugar unit to the two existing cellotriose units.3. Delete overlapping crystal water molecules. These are identified byusing the Subset Interface By_Atom 2.5 command.4. Build hydrogens at a pH of 8.0 and applying charged terminals5. Protonate D121 using the Residue Replace <D121 residue name> ASP Lcommand.6. Apply the CVFF forcefield template through the command PotentialsFix.7. Fix all atoms except the new sugar unit.8. Relax the atomic position of the new sugar unit using 300 cycles ofsimple energy minimization followed by 5000 steps of 1fs simplemolecular dynamics ending by 300 cycles of simple energy minimizationall using the molecular mechanics program Discover 95.0/3.0.1 (Discover95.0/3.0.0 User Guide, October 1995, San Diego: Biosym/MSI, 1995.).

Homology Building of Cellulases

The construction of a structural model of a cellulase with known aminoacid sequence based on a known X-ray structure of the Humicola insolensEGV cellulase consists of the following steps.

1. Define the approximate extend of the core region of the structure tobe modeled and the alignment of the cysteine based on multiple sequencealignment between many known industrially useful cellulase sequences.2. Pair-wise sequence alignment between the new sequence and thesequence of the known X-ray structure.3. Define Structurally Conserved Regions (SCRs) based on the sequencealignment.4. Assign coordinates for the model structure within the SCRs.5. Find structures for the loops or Variable Regions (VRs) between theSCRs by a search in a loop structure database.6. Assign coordinates for the VRs in the model structure from thedatabase search result.7. Create disulfide bonds and set protonation state.8. Refine the build structure using molecular mechanics.

The known X-ray structure of the Humicola insolens EGV cellulase will inthe following be termed the reference structure. The structure to bemodeled will be termed the model structure.

Ad 1: The approximate extent of the core part of the enzyme isdetermined by a multiple sequence alignment including many knowncellulase sequences. Since the reference structure contains only atomiccoordinates for the core part of the enzyme only the residues in thesequence to be modeled which align with the core part of the referencestructure can be included in the model building. This alignment alsodetermines the alignment of the cysteine. The multiple sequencealignment is performed using the Pileup algorithm as described earlier.

Ad 2: A pair-wise sequence alignment is performed as described earlier.If the cysteine in the conserved disulfide bridges and/or the activesite residues (D10 and D121) does not align, a pair-wise sequencealignment using forced pairing of the cysteines in the conserveddisulfide bridges and/or the active site residues is performed asdescribed earlier. The main purpose of the sequence alignment is todefine SCRs (see later) to be used for a model structure generation.

Ad 3: Based on the sequence alignment Structurally Conserved Regions(SCRs) are defined as continuous regions of overlapping sequence with noinsertions or deletions.

Ad 4: Using the computer program Homology 95.0 (Homology User Guide,October 1995. San Diego: Biosym/MSI, 1995.) atomic coordinates in themodel structure can be generated from the atomic coordinates of thereference structure using the command AssignCoords Sequences.

Ad 5: Using the computer program Homology 95.0 possible conformationsfor the remaining regions, named Variable Regions (VRs) are found by asearch in the loop structure database included in Homology 95.0. Thisprocedure is performed for each VR.

Ad 6: If the VR length is smaller than six residues the first loopstructure in the database search result is selected for coordinategeneration. In cases where longer loops are generated the first solutionin the list which does not have severe atomic overlap are selected. Thedegree of atomic overlap can be analyzed using the Bump Monitor AddIntra command in the computer program Insight II 95.0 (Insight II 95.0User Guide, October 1995. San Diego: Biosym/MSI, 1995.) a parameter of0.25 for the Bump command wilt show the severe overlap. If more than tenbumps exists between the inserted loop region and the remaining part ofthe protein the next solution is tested. If no solution is found withthese parameters, the solution with the fewest bumps is selected. Thecoordinates for the VR regions are generated using the commandAssignCoords Loops in the program Homology 95.0.

Ad 7: The disulfide bonds are created using the Bond Create command inthe Biopolymer module of Insight II 95.0 and the protonation state isset to match pH 8.0 with charged caps using the Hydrogens command.Finally the active proton donor (the residue equivalent to D121 in thereference structure) is protonated using the residue replace <D121residue name> ASP L command. To finalize the data of the model theappropriate forcefield template is applied using the CVFF forcefieldthrough the command Potentials Fix.

Ad 8: Finally the modeled structure is subjected to 500 cycles energyminimization using the molecular mechanics program Discover 95.0/3.0.1(Discover 95.0/3.0.0 User Guide, October 1995. San Diego: Biosym/MSI,1995.). The output from the above described procedure is atomiccoordinates describing a structural model for the core domain of a newcellulase based on sequence homology to the Humicola insolens EGVcellulase.

Superpositioning of Cellulase Structures

To overlay two cellulase structures a superposition of the structuresare performed using the Structure Alignment command of the Homology 95.0(Homology User Guide, October 1995. San Diego, Biosym/MSI, 1995.). Allparameters for the command are chosen as the default values.

Determination of Residues within 3 Å and 5 Å from the Substrate

In order to determine the amino acid residues within a specifieddistance from the substrate a given cellulase structure is superimposedon the cellulase part of the model structure of the complex betweenHumicola insolens EGV endoglucanase and celloheptaose as describedabove. The residues within a specified distance of the substrate arethen found using the Interface Subset command of the Insight II 95.0(Insight II 95.0 User Guide, October 1995. San Diego: Biosym/MSI). Thespecified distance is supplied as parameter to the program.

The results of this determination are presented in Tables 2 and 3 below.

Determination of Surface Accessibility

To determine the solvent accessibility the Access_Surf command inHomology 95.0 (Homology User Guide, October 1995: San Diego: Biosym/MSI,1995) was used. The program uses the definition proposed by Lee andRichards (Lee, B. & Richards, F. M. “The interpretation of proteinstructures: Estimation of static accessibility”, J. Mol. Biol., 1971,55, 379-400). A solvent probe radius of 1.4 Å was used and only heavyatoms (i.e., non-hydrogen atoms) were included in the calculation.Residues with zero accessibility are defined as being buried, all otherresidues are defined as being solvent exposed and on the surface of theenzyme structure.

Transferring Level of Specific Activity between Cellulases

In order to transfer the level of catalytic activity between twocellulases, the following protocol is applied using the methodsdescribed above. This method will pinpoint amino acid residuesresponsible for the difference in specific activity, and one or more ofthose amino acid residues must be replaced in one sequence in order totransfer the level of specific activity from the comparison cellulase:

1) Perform multiple sequence alignment of all known industrially usefulcellulases (excluding the Trichoderma reesei cellulases). From thisidentify conserved disulfide bridges amongst the two involved sequencesand the sequence of the Humicola insolens EGV cellulase are identifiedand the active site residues (D10 and D121) are located;2) Perform pair-wise sequence alignment of each sequence with theHumicola insolens EGV cellulase core domain (residues 1-201). If thecysteines in the conserved disulfide bridges do not align at the samepositions and/or if the two active site residues (D10 and D121) do notalign at the same positions then use the pair-wise sequence alignment ofcellulases with forced pairing method. Include only residues in thesequences overlapping with the core domain (residues 1-201) of theHumicola insolens EGV cellulase;3) Create a homology build structure of each sequence;4) Determination of residues within 3 Å from the substrate in each ofthe homology build structures. Differences between the sequences inthese positions will most probably be the residues responsible for thedifference in specific activity. In the case where residues in insertsare found in any of the sequences within the above mentioned distance,the complete insert can be responsible for the difference in specificactivity, and the complete insert must be transferred to the sequencewithout the insert or the complete insert must be deleted in thesequence with the insert;5) If not all specific activity was restored by substitution of residueswithin 3 Å of the substrate determination of residues within 5 Å fromthe substrate in each of the homology build structures will reveal themost probable residues responsible for the remaining difference inspecific activity. In the case where residues in inserts are found inany of the sequences within the above mentioned distance the completeinsert can be responsible for the difference in specific activity, andthe complete insert must be transferred to the sequence without theinsert or the complete insert must be deleted in the sequence with theinsert.Transferring the Level of Stability towards Anionic Tensides betweenCellulases

In order to transfer level of stability towards anionic tensides betweentwo cellulases, the following protocol is applied using the methodsdescribed above. This method will pinpoint amino acid residuesresponsible for the difference in level of stability towards anionictensides, and one or more of those amino acid residues must be replacedin one sequence in order to transfer the level of specific activity fromthe comparison cellulase:

1) Perform multiple sequence alignment of all known industrially usefulcellulases (excluding Trichoderma reesei cellulases). From this identifyconserved disulfide bridges amongst the two involved sequences and thesequence of the Humicola insolens EGV cellulase are identified and theactive site residues (D10 and D121) are located;2) Perform pair-wise sequence alignment of each sequence with theHumicola insolens EGV cellulase core domain (residues 1-201). If thecysteines in the conserved disulfide bridges do not align at the samepositions and/or if the two active site residues (D10 and D121) do notalign at the same positions then use the pair-wise sequence alignment ofcellulases with forced pairing method. Include only residues in thesequences overlapping with the core domain (residues 1-201) of theHumicola insolens EGV cellulase;3) Create a homology build structure of each sequence;4) Determination of residues located at the surface of the enzyme. Thisis done by calculation the surface accessibility. Residues with asurface accessibility greater than 0.0 Å² are exposed to the surface;5) Any residue exposed to the surface belonging to the following groupof amino acids: D, E, H, K, R and C if not involved in a disulfidebridge which differs between the two sequences will most probably beresponsible for the difference in level of stability towards anionictensides. In the case where residues in inserts are found in any of thesequences within the above mentioned group of amino acid types, thecomplete insert can be responsible for the difference in level ofstability towards anionic tensides, and the complete insert must betransferred to the sequence without the insert or the complete insertmust be deleted in the sequence with the insert.

Disulfide Bridges

Disulfide bridges (i.e., Cys-Cys bridges) stabilize the structure of theenzyme. It is believed that a certain number of stabilizing disulfidebridges is necessary to maintain a proper stability of the enzyme.However, it is also contemplated that disulfide bridges can be removedfrom the protein structure resulting in an enzyme variant which is lessstable, especially less thermostable, but which still has significantactivity.

Therefore, in another aspect, the invention provides a cellulase variantwhich variant holds 4 or more of the following disulfide bridges:C11-C135: C12-C47: C16-C86; C31-C56; C87-C199; C89-C189; and C156-C167(cellulase numbering). In a more specific embodiment the variant of theinvention holds 5 or more of the following disulfide bridges: C11-C135;C12-C47; C16-C86: C31-C56; C87-C199; C89-C189; and C156-C167 (cellulasenumbering). In its most specific embodiment, the variant of theinvention holds 6 or more of the following disulfide bridges: C11-C135;C12-C47; C16-C86; C31-C56; C87-C199; C89-C189; and C156-C167 (cellulasenumbering).

In another embodiment the invention provides a cellulase variant inwhich cysteine has been replaced by another natural amino acid at one ormore of the positions 16, 86, 87, 89, 189, and/or 199 (cellulasenumbering).

Binding Cleft Substitutions

In a further aspect, the invention provides a cellulase variant derivedfrom a parental cellulase by substitution, insertion and/or deletion atone or more amino acid residues located in the substrate binding cleft.Mutations introduced at positions close to the substrate affect theenzyme-substrate interactive bindings.

An appropriate way of determining the residues interacting with apotential substrate in a structure is to partitionate the structure in“shells”. The shells are defined as: 1st shell are residues directlyinteracting with the substrate, i.e., closest inter atomic distancebetween substrate and residue both including hydrogen atoms are smallerthan 2.5 Å which will include all direct interaction via hydrogen bondsand other non bonded interactions. The subsequent (2nd, 3rd e.t.c.)shells are defined in the same way, as the residues with inter atomicdistances smaller than 2.5 Å to the substrate or all previouslydetermined shells. In this way the structure will be partitioned inshells. The routine “subset zone” in the program Insight II 95.0(Insight II 95.0 User Guide, October 1995. San Diego: Biosym/MSI, 1995.)can be used to determine the shells.

In a preferred embodiment, the amino acid residue contemplated accordingto this invention is located in the substrate binding cleft at adistance of up to 5 Å from the substrate.

When subjecting the aligned cellulases to the computer modeling methoddisclosed above, the following positions within a distance of up to 5 Åfrom the substrate are revealed: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 18, 18, 19, 20, 21, 21a, 42, 44, 45, 47, 48, 49, 49a, 49b, 74,82, 95j, 110, 111, 112, 113, 114, 115, 116, 119, 121, 123, 127, 128,129, 130, 131, 132, 132a, 133, 145, 146, 147, 148, 149, 150b, 178,and/or 179 (cellulase numbering), cf. Table 2.

Accordingly, in a more specific embodiment, the invention provides acellulase variant which has been derived from a parental cellulase bysubstitution, insertion and/or deletion at one or more of these acidresidues. In a particular embodiment, the cellulase variant is derivedfrom one of the cellulases identified in Table 2 ((a) Humicola insolens;(b) Acremonium sp., (c) Volutella collectotrichoides, (d) Sordariafimicola; (e) Thielavia terrestris; (f) Fusarium oxysporum; (g)Myceliophthora thermophila; (h) Crinipellis scabella; (i) Macrophominaphaseolina, (j) Pseudomonas fluorescens; (k) Ustilago maydis), bysubstitution, insertion and/or deletion at one or more of the positionsidentified in Table 2 for these cellulases.

TABLE 2 Amino Acid Residues less than 5 Å from the Substrate a b c d e fg h i j k   4                   Y   5 S T T S S S T T T A A   6 T T T TT T T T T T T   7 R R R R R R R R R R R   8 Y Y Y Y Y Y Y Y Y Y Y   9 WW W W W W W W W W W  10 D D D D D D D D D D D  11             C  12 C CC C C C C C C C C  13 K K K K K K K K K K L  14 P P P P P P P P P P A 15  S S S S S S S S S H S  16                   C  18 W W W W W W W W WW W  19 A D D S P S P S T   E  20 K E E   G   G G   A  21 K K K K K K KK K   K   21a                   V  42   G T           D  44   V R K Q VR     G  45 S S S S S N S S S S  47 C C C C C C C C C C  48 E D D D N ED D D D S  49   P   49a                     C   49b                    N  74 A A A A A A A A A A F  82 E E E E E E   EE   E   95j                   P 110 S N N S S N N N N N N 111 T T T T TT T T T I V 112 G G G G G G G G G G G 113 G G G G G G G G G Y G 114 D DD D D D D D D D D 115 L L L L L L L L L V V 116         G 119 H H H H QH H H H Q N 121 D D D D D D D D D D D 123   Q 127 G G G G G G G G G G GG 128 G G G G G G G G G G G G 129 V L L V V V V V V V L 130 G G G G G GG G G G G 131 I I I L I I I L I A A 132 F F F F F F F F F F F 132a              T     P 133                 N N 145                   A D146 R R R Q Q Q R Q Q Q Q 147 Y Y Y Y Y Y Y Y Y Y Y 148 G G G G G G G GG G G 149 G     G G G     G 150b                   A 178 D D D D D D D DD D P 179 N N N N N N N N N N V Positions Identified by CellulaseNumbering (a) Humicola insolens; (b) Acremonium sp.; (c) Volutellacollectotrichoides; (d) Sordaria fimicola; (e) Thielavia terrestris; (f)Fusarium oxysporum; (g) Myceliophthora thermophila; (h) Crinipellisscabella; (i) Macrophomina phaseolina; (j) Pseudomonas fluorescens; (k)Ustilago maydis;

In another preferred embodiment, the amino acid residue contemplatedaccording to this invention is located in the substrate binding cleft ata distance of up to 3 Å from the substrate.

When subjecting the aligned cellulases to the computer modeling methoddisclosed above, the following positions within a distance of up to 3 Åfrom the substrate are revealed: 6, 7, 8, 10, 12, 13, 14, 15, 18, 20,21, 45, 48, 74, 110, 111, 112, 113, 114, 115, 119, 121, 127, 128, 129,130, 131, 132, 132a, 146, 147, 148, 150b 178, and/or 179 (cellulasenumbering). cf. Table 3.

Accordingly, in a more specific embodiment, the invention provides acellulase variant which has been derived from a parental cellulase bysubstitution, insertion and/or deletion at one or more of these acidresidues. In a particular embodiment, the cellulase variant is derivedfrom one of the cellulases identified in Table 3 ((a) Humicola insolens;(b) Acremonium sp.; (c) Volutella collectotrichoides; (d) Sordariafimicola; (e) Thielavia terrestris; (f) Fusarium oxysporum, (g)Myceliophthora thermophila; (h) Crinipellis scabella; (i) Macrophominaphaseolina; (j) Pseudomonas fluorescens; (k) Ustilago maydis), bysubstitution, insertion and/or deletion at one or more of the positionsidentified in Table 3 for these cellulases.

TABLE 3 Amino Acid Residues less than 3 Å from the Substrate a b c D e fg h i j K   6 T T T T T T T T T T T   7 R R R R R R R R R R R   8 Y Y YY Y Y Y Y Y Y Y  10 D D D D D D D D D D  12 C C C C C C C C C C C  13 KK K K   K     K   L  14   P     P P P P P   A  15 S S S S S S S S S H S 18 W W W W W W W W W W W  20   E E  21 K     K  45 S S S S S N S S S SS  48   D     N E D     D  74 A     A A A A A A   F 110   N N   S N N NN N N 111 T T T T T T T T T 112 G G G G G G G G G G G 113 G G GG       G G Y G 114 D D D D D D D D D D D 115 L L L L L L L L L V V 119H H H   Q H   H   Q 121 D D D D D D D D D D D 127 G     G G G G   G 128G     G G G G G G   G 129 V L L V V V V V V V L 130 G G G G G G G G G GG 131 I I I L I I I L I A A 132 F F F F F F F F F F F  132a              T     P 146         Q     Q   Q Q 147 Y Y Y Y Y Y Y Y Y YY 148 G G G G G G G G G G G  150b                   A 178 D D DD       D   D P 179 N N N N N N N N N N Positions Identified byCellulase Numbering (a) Humicola insolens; (b) Acremonium sp.; (c)Volutella collectotrichoides; (d) Sordaria fimicola; (e) Thielaviaterrestris; (f) Fusarium oxysporum; (g) Myceliophthora thermophila; (h)Crinipellis scabella; (i) Macrophomina phaseolina; (j) Pseudomonasfluorescens; (k) Ustilago maydis;

Partly Conserved Amino Acid Residues

As defined herein a “partly conserved amino acid residue” is an aminoacid residue identified according to Table 1, at a position at whichposition between 7 to 10 amino acid residues of the 11 residues (i.e.,more than 63%) indicated in Table 1 for that position, are identical.

Accordingly, the invention further provides a cellulase variant, inwhich variant an amino acid residue has been changed into a conservedamino acid residue at one or more positions according to Table 1, atwhich position(s) between 7 and 10 amino acid residues of the 11residues identified in Table 1, are identical.

In a preferred embodiment the invention provides a cellulase variant,which has been derived from a parental cellulase by substitution,insertion and/or deletion at one or more of the following positions: 13,14, 15, 20, 21, 22, 24, 28, 32, 34, 45, 48, 50, 53, 54, 62, 63, 64, 65,66, 68, 69, 70, 71, 72, 73, 74, 75, 79, 85, 88, 90, 92, 93, 95, 96, 97,98, 99, 104, 106, 110, 111, 113, 115, 116, 118, 119, 131, 134, 138, 140,146, 152, 153, 163, 166, 169, 170, 171, 172, 173, 174, 174, 177, 178,179, 180, 193, 196, and/or 197 (cellulase numbering).

In a more specific embodiment the invention provides a cellulase variantthat has been subjected to substitutions, insertions and/or deletions,so as to comprise one or more of the amino acid residues at thepositions identified in Table 4, below. The positions in Table 4reflects the “partly conserved amino acid residue positions” as well asthe non-conserved positions present within 5 Å of the substrate inbinding cleft all of which indeed are present in the aligned sequencesin Table 1.

TABLE 4 Selected Substitutions, Insertions and/or Deletions PositionAmino Acid Residue  4 R, H, K, C, V, Y, M  5 S, T, A 13 K, L 14 P, A 15H, S 16 C, A 19 A D, S, P, T, E 20 A, E G, K 21 K, N  21a V,* 22 A, G, P24 *, L, V 28 A, L, V 32 D, K, N, S 34 D, N 38 F, I, L, Q 42 D, G, T, N,S, K, * 44 K, V, R Q, G, P 45 N, S 46 G, S 47 C, Q 48 D, E, N, S 49 P,S, A, G, *  49a C, *  49b N, * 50 G, N 53 A, G, K, S 54 F, Y 62 F, W 63A, D 64 D, I V 65 D, E, N, S 66 D, N, P, T 68 F, L, P, T, V 69 A, S, T70 L, Y 71 A, G 72 F, W, Y 73 A, G 74 A, F 75 A, G, T, V 79 G, T 82 E, *88 A, G, Q, R 90 F, Y 92 A, L 93 E, Q, T 95 E, T  95j P, * 96 S, T 97 A,G, T 98 A, P 99 L, V 104  L, M 106  F, V 110  N, S 111  I, T, V 113  G,Y 115  L, V 116  G, Q, S 118  G, N, Q, T 119  H, N, Q 129  L, V 131  A,I, L 132  A, P, T, * 133  D, K, N, Q 134  A, G 138  E, Q 145  A, D, N, Q146  Q, R 150b A, * 152  D, S 153  A, K, L, R 163  L, V, W 166  G, S169  F, W 170  F, R 171  A, F, Y 172  D, E, S 173  E, W 174  F, M, W177  A, N 178  D, P 179  N, V 180  L, P 193  I, L 196  I, K, R 197  S, TPositions Identified by Cellulase Numbering

In a yet more preferred embodiment, the invention provides a cellulasevariant derived from a parental cellulase by substitution, insertionand/or deletion at one or more amino acid residues as indicated inTables 5-6, below. The cellulase variant may be derived from anyparental cellulase holding the amino acid residue stated at the positionindicated. In particular the parental cellulase may be a Humicolainsolens cellulase; an Acremonium sp. Cellulase; a Volutellacollectotrichoides cellulase; a Sordaria fimicola cellulase; a Thielaviaterrestris cellulase; a Fusarium oxysporum cellulase; a Myceliophthorathermophila cellulase, a Crinipellis scabella cellulase; a Macrophominaphaseolina cellulase; a Pseudomonas fluorescens cellulase; or a Ustilagomaydis cellulase.

Moreover, the cellulase variant may be characterized by having improvedperformance, in particular with respect to

1. improved performance defined as increased catalytic activity;

2. altered sensitivity to anionic tenside; and/or

3. altered pH optimum;

as also indicated in Tables 5-6. The positions listed in Table 5 reflecttransfer of properties between the different cellulases aligned inTable 1. The positions listed in Table 6 reflect transfer of propertiesfrom Humicola insolens EGV to the other cellulases aligned in Table 1.

TABLE 5 Preferred Cellulase Variants Positions Identified by CellulaseNumbering K13L, L13K (1, 2, 3); P14A, A14P (1); S15H, H15S (1, 3); K20E,K20G, K20A, E20K, G20K, A20K, E20G, E20A, G20E, A20E, G20A, A20G, (1, 2,3); K21N, N21K (1, 2, 3); A22G, A22P, G22A, P22A, G22P, P22G (1); V24*,V24L, *24V, L24V, *24L, L24* (1); V28A, V28L, A28V, L28V, A28L, L28A(1); N32D, N32S, N32K, D32N, S32N, K32N, D32S, D32K, S32D, K32D, S32K,K32S (2, 3); N34D, D34N (2); I38L, I38F, I38Q, L38I, F38I, Q38I, L38F,L38Q, F38L, Q38L, F38Q, Q38F (1) S45N, N45S (1); G46S, S46G (1); E48D,E48N, D48E, N48E, D48N, N48D (1, 2, 3); G50N, N50G (1); A53S, A53G,A53K, S53A, G53A, K53A, S53G, S53K, G53S, K53S, G53K, K53G (1); Y54F,F54Y (1, 3); W62F, F62W (1, 2); A63D, D63A (2, 3); V64I, V64D, I64V,D64V, I64D, D64I (2); N65S, N65D, N65E, S65N, D65N, E65N, S65D, S65E,D65S, E65S, D65E, E65D (2); D66N, D66P, D66T, N66D, P66D, T66D, N66P,N66T, P66N, T66N, P66T, T66P (2, 3); F68V, F68L, F68T, F68P, V68F, L68F,T68F, P68F, V68L, V68T, V68P, L68V, T68V, P68V, L68T, L68P, T68L, P68L,T68P, P68T (1, 2); A69S, A69T, S69A, T69A, S69T, T69S (1); L70Y, Y70L(1); G71A, A71G (1); F72W, F72Y, W72F, Y72F, W72Y, Y72W (1); A73G, G73A(1); A74F, F74A (1); T75V, T75A, T75G, V75T, A75T, G75T, V75A, V75G,A75V, G75V, A75G, G75A (1); G79T, T79G (1); W85T, T85W (1); A88Q, A88G,A88R, Q88A, G88A, R88A, Q88G, Q88R, G88Q, R88Q, G88R, R88G (1, 2, 3);Y90F, F90Y (1); L92A, A92L (1); T93Q, T93E, Q93T, E93T, Q93E, E93Q (2);T95E, E95T (2); S96T, T96S (1); G97T, G97A, T97G, A97G, T97A, A97T (1);P98A, A98P (1); V99L, L99V (1); M104L, L104M (1); V106F, F106V (1, 3);S110N, N110S (1); T111I, T111V, I111T, V111T, I111V, V111I (1); G113Y,Y113G (1, 3); L115V, V115L (1); G116S, G116Q, S116G, Q116G, S116Q, Q116S(1); N118T, N118G, N118Q, T118N, G118N, Q118N, T118G, T118Q, G118T,Q118T, G118Q, Q118G (1); H119Q, H119N, Q119H, N119H (1, 2); V129L, L129V(1); I131L, I131A, L131I, A131I, L131A, A131L (1); G134A, A134G (1);Q138E, E138Q (1, 2, 3); G140N, N140G (1); R146Q, Q146R (1, 2, 3); S152D,D152S (2); R153K, R153L, R153A, K153R, L153R, A153R, K153L, K153A,L153K, A153K, L153A, A153L (2); L163V, L163W, V163L, W163L, V163W, W163V(1); G166S, S166G (1); W169F, F169W (1); R170F, F170R (1, 2, 3); F171Y,F171A, Y171F, A171F, Y171A, A171Y (1); D172E, D172S, E172D, S172D,E172S, S172E (2); W173E, E173W (1, 2, 3); F174M, F174W, M174F, W174F,M174W, W174M (1); A177N, N177A (1); D178P, P178D (1, 2, 3); N179V, V179N(1); P180L, L180P (1); L193I, I193L (1); R196I, R196K, I196R, K196R,I196K, K196I (2, 3); T197S, S197T (1)

TABLE 6 Preferred Cellulase Variants Positions Identified by CellulaseNumbering L13K (1, 2, 3); A14P (1); H15S (1, 3); E20K, G20K, A20K (1, 2,3); N21K (1, 2, 3); G22A, P22A (1); *24V, L24V (1); A28V, L28V (1);D32N, S32N, K32N (2, 3); D34N (2); L38I, F38I, Q38I (1); N45S (1); S46G(1); D48E, N48E (1, 2, 3); N50G (1); S53A, G53A, K53A (1); F54Y (1, 3);F62W (1, 2); D63A (2, 3); I64V, D64V (2); S65N, D65N, E65N (2) N66D,P66D, T66D (2, 3); V68F, L68F, T68F, P68F (1, 2); S69A, T69A (1) Y70L(1) A71G (1) W72F, Y72F (1) G73A (1) F74A (1) V75T, A75T, G75T (1) T79G(1); T85W (1); Q88A, G88A, R88A (1, 2, 3) F90Y (1) A92L (1) Q93T, E93T(2); E95T (2); T96S (1); T97G, A97G (1); A98P (1); L99V (1); L104M (1);F106V (1, 3); N110S (1); I111T, V111T (1); Y113G (1, 3); V115L (1);S116G, Q116G (1); T118N, G118N, Q118N (1); Q119H, N119H (1, 2); L129V(1); L131I, A131I (1); A134G (1); E138Q (1, 2, 3); N140G (1); Q146R (1,2, 3); D152S (2); K153R, L153R, A153R (2); V163L, W163L (1); S166G (1);F169W (1); F170R (1, 2, 3); Y171F, A171F (1); E172D, S172D (2); E173W(1, 2, 3); M174F, W174F (1); N177A (1); P178D (1, 2, 3); V179N (1);L180P (1); I193L (1); I196R, K196R (2, 3); S197T (1)

Altered Sensibility Towards Anionic Tensides

As mentioned above, anionic tensides are products frequentlyincorporated into detergent compositions. Sometimes cellulolytic enzymeshaving an increased stability towards anionic tensides are desired, andsometimes cellulolytic enzymes having an increased sensitivity arepreferred. In a further aspect the invention provides cellulase variantshaving an altered anionic tenside sensitivity.

Accordingly, a cellulase variant of the invention of altered anionictenside sensitivity is a cellulase variant which has been derived from aparental cellulase by substitution, insertion and/or deletion at one ormore of the following positions, 2, 4, 7, 8, 10, 13, 15, 19, 20, 21, 25,26, 29, 32, 33, 34, 35, 37, 40, 42, 42a, 43, 44, 48, 53, 54, 55, 58, 59,68, 64, 65, 66, 67, 70, 72, 76, 79, 80, 82, 84, 86, 88, 90, 91, 93, 95,95j, 95, 95j, 97, 100, 101, 102, 103, 113, 114, 117, 119, 121, 133, 136,137, 138, 139, 140a, 141, 143a, 145, 146, 147, 150e, 150j, 151, 152,153, 154, 155, 156, 157, 158, 159, 160c, 160e, 160k, 161, 162, 164, 165,168, 170, 171, 172, 173, 175, 176, 178, 181, 183, 184, 185, 186, 188,191, 192, 195, 196, 200, and/or 201 (cellulase numbering). Thesepositions contain, in at least one of the cellulase sequences aligned inTable 1, a charged or potentially charged aa residue.

In a particular embodiment, the cellulase variant is derived from one ofthe cellulases identified in Table 7, below, ((a) Humicola insolens, (b)Acremonium sp., (c) Volutella collectotrichoides; (d) Sordaria fimicola;(e) Thielavia terrestris; (f) Fusarium oxysporum; (g) Myceliophthorathermophila; (h) Crinipellis scabella; (i) Macrophomina phaseolina; (j)Pseudomonas fluorescens, (k) Ustilago maydis), by substitution,insertion and/or deletion at one or more of the positions identified inTable 7 for these cellulases.

TABLE 7 Altered Sensitivity towards Anionic Tensides a b c d e f g h i jk   2 D   4 R H R K   H       Y   7 R R R R R R R R R R R   8 Y Y Y Y YY Y Y Y Y Y  10 D D D D D D D D D D D  13 K K K K K K K K K K  15                  H  19   D D               E  20 K E E  21 K K K K K KK K K K  25                     Y  26 R R K  29     K   Y     R     D 32   D D D D D D D D   K  33   R R     K K R  34                     D 35           D D   D  37 R       R         R  40 D     D D   D     D D 42 D               D     K   42a               D     K  43                    D  44 K   R K     R K K  48 E D D D   E D D D D  53                    K  54 Y   Y Y Y Y Y   Y Y  55                 Y  58D D D   D         D  59           Y       K  63                   D  64                  D  65               D     E  66 D   D D D D D   D  67D         E E       D  70   Y Y Y Y Y Y Y Y Y  72                   Y 76       K   K K H K  79                   D  80                 K  82E E E E E E E E E   E  84                   D   D  86                    D  88                   R  90 Y Y Y Y Y Y Y Y Y Y 91 E           E E K   Y  93                     E  95                    E   95d                   Y   95h                    H   95i                   D D  97                    K 100           K         K 101                    R 102 K K K K K K K K K K 103 K         K K K     K113                   Y 114 D D D D D D D D D D D 117           D D 119H H H H   H H H H 121 D D D D D D D D D D D 133 D D D D   D         K136       K 137        R     D   K 138        E   E 139             Y140a           K 141                     E 143a                   E L145             D       D 146 R R R       R 147 Y Y Y Y Y Y Y Y Y Y Y150e                   K 150j                   Y 151            H       K 152                 D 153 R R R R R R K R R 154        D   E D 155 E     E   E E       E 156                   Y 157D     D D D E     K 158 R E                 K 159           Y  160c                  R  160e                   D  160k                   R161 D         E E       K 162                 K 164 K R   K K K K 165  D       D         E 168 Y H       H         K 170 R R R R R R R RR   R 171   Y Y 172 D D D D D D D D D E 173                     E 175K     K   E     E E 176   D D               D 178 D D D D D D D D D D181     D E   D         K 183                 D K 184                  Y 185 R R R K   E   R E K K 186           RR       E     E R 188   R                 K 191           K         K192 E     E E   E   E E 195     D     D         D 196 R R R R R   K   RR R 200 R   R K K K 201 R R R R R R R R R R R Positions Identified byCellulase Numbering (a) Humicola insolens; (b) Acremonium sp.; (c)Volutella collectotrichoides; (d) Sordaria fimicola; (e) Thielaviaterrestris; (f) Fusarium oxysporum; (g) Myceliophthora thermophila; (h)Crinipellis scabella; (i) Macrophomina phaseolina; (j) Pseudomonasfluorescens; (k) Ustilago maydis;

Enzyme Compositions

In a still further aspect, the present invention relates to an enzymecomposition comprising an enzyme exhibiting cellulolytic activity asdescribed above.

The enzyme composition of the invention may, in addition to thecellulase of the invention, comprise one or more other enzyme types, forinstance hemi-cellulase such as xylanase and mannanase, other cellulasecomponents, chitinase, lipase, esterase, pectinase, cutinase, phytase,oxidoreductase, peroxidase, laccase, oxidase, pactinmethylesterase,polygalacturonase, protease, or amylase.

The enzyme composition may be prepared in accordance with methods knownin the art and may be in the form of a liquid or a dry composition. Forinstance, the enzyme composition may be in the form of a granulate or amicrogranulate. The enzyme to be included in the composition may bestabilized in accordance with methods known in the art.

Examples are given below of preferred uses of the enzyme composition ofthe invention. The dosage of the enzyme composition of the invention andother conditions under which the composition is used may be determinedon the basis of methods known in the art.

The enzyme composition according to the invention may be useful for atleast one of the following purposes.

Uses

During washing and wearing, dyestuff from dyed fabrics or garment willconventionally bleed from the fabric which then looks faded and worn.Removal of surface fibers from the fabric will partly restore theoriginal colors and looks of the fabric. By the term “colorclarification”, as used herein, is meant the partly restoration of theinitial colors of fabric or garment throughout multiple washing cycles.

The term “de-pilling” denotes removing of pills from the fabric surface.

The term “soaking liquor” denotes an aqueous liquor in which laundry maybe immersed prior to being subjected to a conventional washing process.The soaking liquor may contain one or more ingredients conventionallyused in a washing or laundering process.

The term “washing liquor” denotes an aqueous liquor in which laundry issubjected to a washing process, i.e., usually a combined chemical andmechanical action either manually or in a washing machine.Conventionally, the washing liquor is an aqueous solution of a powder orliquid detergent composition.

The term “rinsing liquor” denotes an aqueous liquor in which laundry isimmersed and treated, conventionally immediately after being subjectedto a washing process, in order to rinse the laundry, i.e. essentiallyremove the detergent solution from the laundry. The rinsing liquor maycontain a fabric conditioning or softening composition.

The laundry subjected to the method of the present invention may beconventional washable laundry. Preferably, the major part of the laundryis sewn or un-sewn fabrics, including knits, wovens, denims, yarns, andtoweling, made from cotton, cotton blends or natural or manmadecellulosics (e.g., originating from xylan-containing cellulose fiberssuch as from wood pulp) or blends thereof. Examples of blends are blendsof cotton or rayon/viscose with one or more companion material such aswool, synthetic fibers (e.g., polyamide fibers, acrylic fibers,polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers,polyvinylidene chloride fibers, polyurethane fibers, polyurea fibers,aramid fibers), and cellulose-containing fibers (e.g., rayon/viscose,ramie, flax/linen, jute, cellulose acetate fibers, lyocell).

Detergent Disclosure and Examples Surfactant System

The detergent compositions according to the present invention comprise asurfactant system, wherein the surfactant can be selected from nonionicand/or anionic and/or cationic and/or ampholytic and/or zwitterionicand/or semi-polar surfactants.

The surfactant is typically present at a level from 0.1% to 60% byweight.

The surfactant is preferably formulated to be compatible with enzymecomponents present in the composition. In liquid or gel compositions thesurfactant is most preferably formulated in such a way that it promotes,or at least does not degrade, the stability of any enzyme in thesecompositions.

Preferred systems to be used according to the present invention compriseas a surfactant one or more of the nonionic and/or anionic surfactantsdescribed herein.

Polyethylene, polypropylene, and polybutylene oxide condensates of alkylphenols are suitable for use as the nonionic surfactant of thesurfactant systems of the present invention, with the polyethylene oxidecondensates being preferred. These compounds include the condensationproducts of alkyl phenols having an alkyl group containing from about 6to about 14 carbon atoms, preferably from about 8 to about 14 carbonatoms, in either a straight chain or branched-chain configuration withthe alkylene oxide. In a preferred embodiment, the ethylene oxide ispresent in an amount equal to from about 2 to about 25 moles, morepreferably from about 3 to about 15 moles, of ethylene oxide per mole ofalkyl phenol. Commercially available nonionic surfactants of this typeinclude Igepal™ CO-630, marketed by the GAF Corporation; and Triton™X-45, X-114, X-100 and X-102, all marketed by the Rohm & Haas Company.These surfactants are commonly referred to as alkylphenol alkoxylates(e.g., alkyl phenol ethoxylates).

The condensation products of primary and secondary aliphatic alcoholswith about 1 to about 25 moles of ethylene oxide are suitable for use asthe nonionic surfactant of the nonionic surfactant systems of thepresent invention. The alkyl chain of the aliphatic alcohol can eitherbe straight or branched, primary or secondary, and generally containsfrom about 8 to about 22 carbon atoms. Preferred are the condensationproducts of alcohols having an alkyl group containing from about 8 toabout 20 carbon atoms, more preferably from about 10 to about 18 carbonatoms, with from about 2 to about 10 moles of ethylene oxide per mole ofalcohol. About 2 to about 7 moles of ethylene oxide and most preferablyfrom 2 to 5 moles of ethylene oxide per mole of alcohol are present insaid condensation products. Examples of commercially available nonionicsurfactants of this type include Tergitol™ 15-S-9 (The condensationproduct of C11-C15 linear alcohol with 9 moles ethylene oxide),Tergitol™ 24-L-6 NMW (the condensation product of C12-C14 primaryalcohol with 6 moles ethylene oxide with a narrow molecular weightdistribution), both marketed by Union Carbide Corporation, Neodol™ 45-9(the condensation product of C14-C15 linear alcohol with 9 moles ofethylene oxide), Neodol™ 23-3 (the condensation product of C12-C13linear alcohol with 3 moles of ethylene oxide), Neodol™ 45-7 (thecondensation product of C14-C15 linear alcohol with 7 moles of ethyleneoxide), Neodol™ 45-5 (the condensation product of C14-C15 linear alcoholwith 5 moles of ethylene oxide) marketed by Shell Chemical Company,Kyro™ EOB (the condensation product of C13-C15 alcohol with 9 molesethylene oxide), marketed by The Procter & Gamble Company, and GenapolLA 050 (the condensation product of C12-C14 alcohol with 5 moles ofethylene oxide) marketed by Hoechst. Preferred range of HLB in theseproducts is from 8-11 and most preferred from 8-10.

Also useful as the nonionic surfactant of the surfactant systems of thepresent invention are alkylpolysaccharides disclosed in U.S. Pat. No.4,565,647, having a hydrophobic group containing from about 6 to about30 carbon atoms, preferably from about 10 to about 16 carbon atoms and apolysaccharide, e.g., a polyglycoside, hydrophilic group containing fromabout 1.3 to about 10, preferably from about 1.3 to about 3, mostpreferably from about 1.3 to about 2.7 saccharide units. Any reducingsaccharide containing 5 or 6 carbon atoms can be used, e.g., glucose,galactose and galactosyl moieties can be substituted for the glucosylmoieties (optionally the hydrophobic group is attached at the 2-, 3-,4-, etc. positions thus giving a glucose or galactose as opposed to aglucoside or galactoside). The intersaccharide bonds can be, e.g.,between the one position of the additional saccharide units and the 2-,3-, 4-, and/or 6-positions on the preceding saccharide units.

The preferred alkylpolyglycosides have the formula

R2O(CnH₂nO)t(glycosyl)x

wherein R2 is selected from the group consisting of alkyl, alkylphenyl,hydroxyalkyl, hydroxyalkylphenyl, and mixtures thereof in which thealkyl groups contain from about 10 to about 18, preferably from about 12to about 14, carbon atoms; n is 2 or 3, preferably 2; t is from 0 toabout 10, preferably 0; and x is from about 1.3 to about 10, preferablyfrom about 1.3 to about 3, most preferably from about 1.3 to about 2.7.The glycosyl is preferably derived from glucose. To prepare thesecompounds, the alcohol or alkylpolyethoxy alcohol is formed first andthen reacted with glucose, or a source of glucose, to form the glucoside(attachment at the 1-position). The additional glycosyl units can thenbe attached between their 1-position and the preceding glycosyl units2-, 3-, 4-, and/or 6-position, preferably predominantly the 2-position.

The condensation products of ethylene oxide with a hydrophobic baseformed by the condensation of propylene oxide with propylene glycol arealso suitable for use as the additional nonionic surfactant systems ofthe present invention. The hydrophobic portion of these compounds willpreferably have a molecular weight from about 1500 to about 1800 andwill exhibit water insolubility. The addition of polyoxyethylenemoieties to this hydrophobic portion tends to increase the watersolubility of the molecule as a whole, and the liquid character of theproduct is retained up to the point where the polyoxyethylene content isabout 50% of the total weight of the condensation product, whichcorresponds to condensation with up to about 40 moles of ethylene oxide.Examples of compounds of this type include certain of the commerciallyavailable Pluronic™ surfactants, marketed by BASF.

Also suitable for use as the nonionic surfactant of the nonionicsurfactant system of the present invention, are the condensationproducts of ethylene oxide with the product resulting from the reactionof propylene oxide and ethylenediamine. The hydrophobic moiety of theseproducts consists of the reaction product of ethylenediamine and excesspropylene oxide, and generally has a molecular weight of from about 2500to about 3000. This hydrophobic moiety is condensed with ethylene oxideto the extent that the condensation product contains from about 40% toabout 80% by weight of polyoxyethylene and has a molecular weight offrom about 5,000 to about 11,000. Examples of this type of nonionicsurfactant include certain of the commercially available Tetronic™compounds, marketed by BASF.

Preferred for use as the nonionic surfactant of the surfactant systemsof the present invention are polyethylene oxide condensates of alkylphenols, condensation products of primary and secondary aliphaticalcohols with from about 1 to about 25 moles of ethyleneoxide,alkylpolysaccharides, and mixtures hereof. Most preferred are C8-C14alkyl phenol ethoxylates having from 3 to 15 ethoxy groups and C8-C18alcohol ethoxylates (preferably C10 avg.) having from 2 to 10 ethoxygroups, and mixtures thereof.

Highly preferred nonionic surfactants are polyhydroxy fatty acid amidesurfactants of the formula

wherein R1 is H, or R1 is C1-4 hydrocarbyl, 2-hydroxyethyl,2-hydroxypropyl or a mixture thereof, R2 is C5-31 hydrocarbyl, and Z isa polyhydroxyhydrocarbyl having a linear hydrocarbyl chain with at least3 hydroxyls directly connected to the chain, or an alkoxylatedderivative thereof. Preferably, R1 is methyl, R2 is straight C11-15alkyl or C16-18 alkyl or alkenyl chain such as coconut alkyl or mixturesthereof, and Z is derived from a reducing sugar such as glucose,fructose, maltose or lactose, in a reductive amination reaction.

Highly preferred anionic surfactants include alkyl alkoxylated sulfatesurfactants. Examples hereof are water soluble salts or acids of theformula RO(A)mSO3M wherein R is an unsubstituted C10-C-24 alkyl orhydroxyalkyl group having a C10-C24 alkyl component, preferably aC12-C20 alkyl or hydro-xyalkyl, more preferably C12-C18 alkyl orhydroxyalkyl, A is an ethoxy or propoxy unit, m is greater than zero,typically between about 0.5 and about 6, more preferably between about0.5 and about 3, and M is H or a cation which can be, for example, ametal cation (e.g., sodium, potassium, lithium, calcium, magnesium,etc.), ammonium or substituted-ammonium cation. Alkyl ethoxylatedsulfates as well as alkyl propoxylated sulfates are contemplated herein.Specific examples of substituted ammonium cations include methyl-,dimethyl, trimethyl-ammonium cations and quaternary ammonium cationssuch as tetramethyl-ammonium and dimethyl piperidinium cations and thosederived from alkylamines such as ethylamine, diethylamine,triethylamine, mixtures thereof, and the like. Exemplary surfactants areC12-C18 alkyl polyethoxylate (1.0) sulfate (C12-C18E(1.0)M), C12-C18alkyl polyethoxylate (2.25) sulfate (C12-C18(2.25)M, and C12-C18 alkylpolyethoxylate (3.0) sulfate (C12-C18E(3.0)M), and C12-C18 alkylpolyethoxylate (4.0) sulfate (C12-C18E(4.0)M), wherein M is convenientlyselected from sodium and potassium.

Suitable anionic surfactants to be used are alkyl ester sulfonatesurfactants including linear esters of C8-C20 carboxylic acids (i.e.,fatty acids) which are sulfonated with gaseous SO₃ according to “TheJournal of the American Oil Chemists Society”, 52, 1975, pp. 323-329.Suitable starting materials would include natural fatty substances asderived from tallow, palm oil, etc.

The preferred alkyl ester sulfonate surfactant, especially for laundryapplications, comprises alkyl ester sulfonate surfactant of thestructural formula:

wherein R3 is a C8-C20 hydrocarbyl, preferably an alkyl, or combinationthereof, R4 is a C1-C6 hydrocarbyl, preferably an alkyl, or combinationthereof, and M is a cation which forms a water soluble salt with thealkyl ester sulfonate. Suitable salt-forming cations include metals suchas sodium, potassium, and lithium, and substituted or unsubstitutedammonium cations, such as monoethanolamine, diethonolamine, andtriethanolamine. Preferably, R3 is C10-C16 alkyl, and R4 is methyl,ethyl or isopropyl. Especially preferred are the methyl ester sulfonateswherein R3 is C10-C16 alkyl.

Other suitable anionic surfactants include the alkyl sulfate surfactantswhich are water soluble salts or acids of the formula ROSO₃M wherein Rpreferably is a C10-C24 hydrocarbyl, preferably an alkyl or hydroxyalkylhaving a C10-C20 alkyl component, more preferably a C12-C18 alkyl orhydroxyalkyl, and M is H or a cation, e.g., an alkali metal cation(e.g., sodium, potassium, lithium), or ammonium or substituted ammonium(e.g., methyl-, dimethyl-, and trimethyl ammonium cations and quaternaryammonium cations such as tetramethyl-ammonium and dimethyl piperidiniumcations and quaternary ammonium cations derived from alkylamines such asethylamine, diethylamine, triethylamine, and mixtures thereof, and thelike). Typically, alkyl chains of C12-C16 are preferred for lower washtemperatures (e.g., below about 50° C.) and C16-C18 alkyl chains arepreferred for higher wash temperatures (e.g., above about 50° C.).

Other anionic surfactants useful for detersive purposes can also beincluded in the laundry detergent compositions of the present invention.Theses can include salts (including, for example, sodium, potassium,ammonium, and substituted ammonium salts such as mono- di- andtriethanolamine salts) of soap, C8-C22 primary or secondaryalkanesulfonates, C8-C24 olefinsulfonates, sulfonated polycarboxylicacids prepared by sulfonation of the pyrolyzed product of alkaline earthmetal citrates, e.g., as described in British patent specification No.1,082,179, C8-C24 alkylpolyglycolethersulfates (containing up to 10moles of ethylene oxide); alkyl glycerol sulfonates, fatty acyl glycerolsulfonates, fatty oleyl glycerol sulfates, alkyl phenol ethylene oxideether sulfates, paraffin sulfonates, alkyl phosphates, isethionates suchas the acyl isethionates, N-acyl taurates, alkyl succinamates andsulfosuccinates, monoesters of sulfosuccinates (especially saturated andunsaturated C12-C18 monoesters) and diesters of sulfosuccinates(especially saturated and unsaturated C6-C12 diesters), acylsarcosinates, sulfates of alkylpolysaccharides such as the sulfates ofalkylpolyglucoside (the nonionic nonsulfated compounds being describedbelow), branched primary alkyl sulfates, and alkyl polyethoxycarboxylates such as those of the formula RO(CH₂CH₂O)k—CH₂C00-M+ whereinR is a C8-C22 alkyl, k is an integer from 1 to 10, and M is a solublesalt forming cation. Resin acids and hydrogenated resin acids are alsosuitable, such as rosin, hydrogenated rosin, and resin acids andhydrogenated resin acids present in or derived from tall oil.

Alkylbenzene sulfonates are highly preferred. Especially preferred arelinear (straight-chain) alkyl benzene sulfonates (LAS) wherein the alkylgroup preferably contains from 10 to 18 carbon atoms.

Further examples are described in “Surface Active Agents and Detergents”(Vols. I and II by Schwartz, Perrry and Berch). A variety of suchsurfactants are also generally disclosed in U.S. Pat. No. 3,929,678(column 23, line 58 through column 29, line 23, herein incorporated byreference).

When included therein, the laundry detergent compositions of the presentinvention typically comprise from about 1% to about 40%, preferably fromabout 3% to about 20% by weight of such anionic surfactants.

The laundry detergent compositions of the present invention may alsocontain cationic, ampholytic, zwitterionic, and semi-polar surfactants,as well as the nonionic and/or anionic surfactants other than thosealready described herein.

Cationic detersive surfactants suitable for use in the laundry detergentcompositions of the present invention are those having one long-chainhydrocarbyl group. Examples of such cationic surfactants include theammonium surfactants such as alkyltrimethylammonium halogenides, andthose surfactants having the formula:

[R2(OR3)y][R4(OR3)y]2R5N+X−

wherein R2 is an alkyl or alkyl benzyl group having from about 8 toabout 18 carbon atoms in the alkyl chain, each R3 is selected form thegroup consisting of —CH₂CH₂—, —CH₂CH(CH₃)—, —CH₂CH(CH₂OH)—, —CH₂CH₂CH₂—,and mixtures thereof; each R4 is selected from the group consisting ofC1-C4 alkyl, C1-C4 hydroxyalkyl, benzyl ring structures formed byjoining the two R4 groups, —CH₂CHOHCHOHCOR6CHOHCH₂OH, wherein R6 is anyhexose or hexose polymer having a molecular weight less than about 1000,and hydrogen when y is not 0; R5 is the same as R4 or is an alkyl chain,wherein the total number of carbon atoms or R2 plus R5 is not more thanabout 18; each y is from 0 to about 10, and the sum of the y values isfrom 0 to about 15; and X is any compatible anion.

Highly preferred cationic surfactants are the water soluble quaternaryammonium compounds useful in the present composition having the formula:

R1R2R3R4N+X−  (i)

wherein R1 is C8-C16 alkyl, each of R2, R3 and R4 is independently C1-C4alkyl, C1-C4 hydroxy alkyl, benzyl, and —(C2H40)xH where x has a valuefrom 2 to 5 and X is an anion. Not more than one of R2, R3 or R4 shouldbe benzyl.

The preferred alkyl chain length for R1 is C12-C15, particularly wherethe alkyl group is a mixture of chain lengths derived from coconut orpalm kernel fat or is derived synthetically by olefin build up or OXOalcohols synthesis.

Preferred groups for R2, R3 and R4 are methyl and hydroxyethyl groupsand the anion X may be selected from halide, methosulphate, acetate andphosphate ions.

Examples of suitable quaternary ammonium compounds of formulae (i) foruse herein are:

coconut trimethyl ammonium chloride or bromide;

coconut methyl dihydroxyethyl ammonium chloride or bromide;

decyl triethyl ammonium chloride;

decyl dimethyl hydroxyethyl ammonium chloride or bromide;

C12-15 dimethyl hydroxyethyl ammonium chloride or bromide, coconutdimethyl hydroxyethyl ammonium chloride or bromide, myristyl trimethylammonium methyl sulphate;

lauryl dimethyl benzyl ammonium chloride or bromide;

lauryl dimethyl (ethenoxy)4 ammonium chloride or bromide;

choline esters (compounds of formula (i) wherein R1 is

alkyl and R2R3R4 are methyl).

di-alkyl imidazolines [compounds of formula (i)].

Other cationic surfactants useful herein are also described in U.S. Pat.No. 4,228,044 and in EP 000 224.

When included therein, the laundry detergent compositions of the presentinvention typically comprise from 0.2% to about 25%, preferably fromabout 1% to about 8% by weight of such cationic surfactants.

Ampholytic surfactants are also suitable for use in the laundrydetergent compositions of the present invention. These surfactants canbe broadly described as aliphatic derivatives of secondary or tertiaryamines, or aliphatic derivatives of heterocyclic secondary and tertiaryamines in which the aliphatic radical can be straight or branched-chain.One of the aliphatic substituents contains at least about 8 carbonatoms, typically from about 8 to about 18 carbon atoms, and at least onecontains an anionic water-solubilizing group, e.g., carboxy, sulfonate,sulfate. See U.S. Pat. No. 3,929,678 (column 19, lines 18-35) forexamples of ampholytic surfactants.

When included therein, the laundry detergent compositions of the presentinvention typically comprise from 0.2% to about 15%, preferably fromabout 1% to about 10% by weight of such ampholytic surfactants.

Zwitterionic surfactants are also suitable for use in laundry detergentcompositions. These surfactants can be broadly described as derivativesof secondary and tertiary amines, derivatives of heterocyclic secondaryand tertiary amines, or derivatives of quaternary ammonium, quaternaryphosphonium or tertiary sulfonium compounds. See U.S. Pat. No. 3,929,678(column 19, line 38 through column 22, line 48) for examples ofzwitterionic surfactants.

When included therein, the laundry detergent compositions of the presentinvention typically comprise from 0.2% to about 15%, preferably fromabout 1% to about 10% by weight of such zwitterionic surfactants.

Semi-polar nonionic surfactants are a special category of nonionicsurfactants which include water-soluble amine oxides containing onealkyl moiety of from about 10 to about 18 carbon atoms and 2 moietiesselected from the group consisting of alkyl groups and hydroxyalkylgroups containing from about 1 to about 3 carbon atoms; water solublephosphine oxides containing one alkyl moiety of from about 10 to about18 carbon atoms and 2 moieties selected from the group consisting ofalkyl groups and hydroxyalkyl groups containing from about 1 to about 3carbon atoms; and water-soluble sulfoxides containing one alkyl moietyfrom about 10 to about 18 carbon atoms and a moiety selected from thegroup consisting of alkyl and hydroxyalkyl moieties of from about 1 toabout 3 carbon atoms.

Semi-polar nonionic detergent surfactants include the amine oxidesurfactants having the formula:

wherein R3 is an alkyl, hydroxyalkyl, or alkyl phenyl group or mixturesthereof containing from about 8 to about 22 carbon atoms; R4 is analkylene or hydroxyalkylene group containing from about 2 to about 3carbon atoms or mixtures thereof: x is from 0 to about 3: and each R5 isan alkyl or hydroxyalkyl group containing from about 1 to about 3 carbonatoms or a polyethylene oxide group containing from about 1 to about 3ethylene oxide groups. The R5 groups can be attached to each other,e.g., through an oxygen or nitrogen atom, to form a ring structure.

These amine oxide surfactants in particular include C10-C18 alkyldimethyl amine oxides and C8-C12 alkoxy ethyl dihydroxy ethyl amineoxides.

When included therein, the laundry detergent compositions of the presentinvention typically comprise from 0.2% to about 15%, preferably fromabout 1% to about 10% by weight of such semi-polar nonionic surfactants.

Builder System

The compositions according to the present invention may further comprisea builder system. Any conventional builder system is suitable for useherein including aluminosilicate materials, silicates, polycarboxylatesand fatty acids, materials such as ethylenediamine tetraacetate, metalion sequestrants such as aminopolyphosphonates, particularlyethylenediamine tetramethylene phosphonic acid and diethylene triaminepentamethylenephosphonic acid. Though less preferred for obviousenvironmental reasons, phosphate builders can also be used herein.

Suitable builders can be an inorganic ion exchange material, commonly aninorganic hydrated aluminosilicate material, more particularly ahydrated synthetic zeolite such as hydrated zeolite A, X, B, HS or MAP.

Another suitable inorganic builder material is layered silicate, e.g.,SKS-6 (Hoechst). SKS-6 is a crystalline layered silicate consisting ofsodium silicate (Na₂Si₂O₅).

Suitable polycarboxylates containing one carboxy group include lacticacid, glycolic acid and ether derivatives thereof as disclosed inBelgian Patent Nos. 831,368, 821,369 and 821,370. Polycarboxylatescontaining two carboxy groups include the water-soluble salts ofsuccinic acid, malonic acid, (ethylenedioxy) diacetic acid, maleic acid,diglycollic acid, tartaric acid, tartronic acid and fumaric acid, aswell as the ether carboxylates described in German Offenle-enschrift2,446,686, and 2,446,487, U.S. Pat. No. 3,935,257 and the sulfinylcarboxylates described in Belgian Patent No. 840,623. Polycarboxylatescontaining three carboxy groups include, in particular, water-solublecitrates, aconitrates and citraconates as well as succinate derivativessuch as the carboxymethyloxysuccinates described in British Patent No.1,379,241 lactoxysuccinates described in Netherlands Application7205873, and the oxypolycarboxylate materials such as2-oxa-1,1,3-propane tricarboxylates described in British Patent No.1,387,447.

Polycarboxylates containing four carboxy groups include oxydisuccinatesdisclosed in British Patent No. 1,261,829, 1,1,2,2,-ethanetetracarboxylates, 1,1,3,3-propane tetracarboxylates containing sulfosubstituents include the sulfosuccinate derivatives disclosed in BritishPatent Nos. 1,398,421 and 1,398,422 and in U.S. Pat. No. 3,936,448, andthe sulfonated pyrolysed citrates described in British Patent No.1,082,179, while polycarboxylates containing phosphone substituents aredisclosed in British Patent No. 1,439,000.

Alicyclic and heterocyclic polycarboxylates include cyclopentane-cis,cis-cis-tetracarboxylates, cyclopentadienide pentacarboxylates,2,3,4,5-tetrahydro-furan-cis, cis, cis-tetracarboxylates,2,5-tetrahydro-furan-cis, discarboxylates,2,2,5,5,-tetrahydrofuran-tetracarboxylates,1,2,3,4,5,6-hexane-hexacarboxylates and carboxymethyl derivatives ofpolyhydric alcohols such as sorbitol, mannitol and xylitol. Aromaticpolycarboxylates include mellitic acid, pyromellitic acid and thephthalic acid derivatives disclosed in British Patent No. 1,425,343.

Of the above, the preferred polycarboxylates are hydroxy-carboxylatescontaining up to three carboxy groups per molecule, more particularlycitrates.

Preferred builder systems for use in the present compositions include amixture of a water-insoluble aluminosilicate builder such as zeolite Aor of a layered silicate (SKS-6), and a water-soluble carboxylatechelating agent such as citric acid.

A suitable chelant for inclusion in the detergent compositions inaccordance with the invention is ethylenediamine-N,N′-disuccinic acid(EDDS) or the alkali metal, alkaline earth metal, ammonium, orsubstituted ammonium salts thereof, or mixtures thereof. Preferred EDDScompounds are the free acid form and the sodium or magnesium saltthereof. Examples of such preferred sodium salts of EDDS include Na₂EDDSand Na₄EDDS. Examples of such preferred magnesium salts of EDDS includeMgEDDS and Mg₂EDDS. The magnesium salts are the most preferred forinclusion in compositions in accordance with the invention.

Preferred builder systems include a mixture of a water-insolublealuminosilicate builder such as zeolite A, and a water solublecarboxylate chelating agent such as citric acid.

Other builder materials that can form part of the builder system for usein granular compositions include inorganic materials such as alkalimetal carbonates, bicarbonates, silicates, and organic materials such asthe organic phosphonates, amino polyalkylene phosphonates and aminopolycarboxylates.

Other suitable water-soluble organic salts are the homo- or co-polymericacids or their salts, in which the polycarboxylic acid comprises atleast two carboxyl radicals separated form each other by not more thantwo carbon atoms.

Polymers of this type are disclosed in GB-A-1,596,756. Examples of suchsalts are polyacrylates of MW 2000-5000 and their copolymers with maleicanhydride, such copolymers having a molecular weight of from 20,000 to70,000, especially about 40,000.

Detergency builder salts are normally included in amounts of from 5% to80% by weight of the composition. Preferred levels of builder for liquiddetergents are from 5% to 30%.

Enzymes

Preferred detergent compositions, in addition to the enzyme preparationof the invention, comprise other enzyme(s) which provides cleaningperformance and/or fabric care benefits.

Such enzymes include proteases, lipases, cutinases, amylases,cellulases, peroxidases, oxidases (e.g., laccases).

Proteases: Any protease suitable for use in alkaline solutions can beused. Suitable proteases include those of animal, vegetable or microbialorigin. Microbial origin is preferred. Chemically or geneticallymodified mutants are included. The protease may be a serine protease,preferably an alkaline microbial protease or a trypsin-like protease.Examples of alkaline proteases are subtilisins, especially those derivedfrom Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin309, subtilisin 147 and subtilisin 168 (described in WO 89/06279).Examples of trypsin-like proteases are trypsin (e.g., of porcine orbovine origin) and the Fusarium protease described in WO 89/06270.

Preferred commercially available protease enzymes include those soldunder the trade names Alcalase, Savinase, Primase, Durazym, and Esperaseby Novo Nordisk A/S (Denmark), those sold under the tradename Maxatase,Maxacal, Maxapem, Properase, Purafect and Purafect OXP by GenencorInternational, and those sold under the tradename Opticlean and Optimaseby Solvay Enzymes. Protease enzymes may be incorporated into thecompositions in accordance with the invention at a level of from0.00001% to 2% of enzyme protein by weight of the composition,preferably at a level of from 0.0001% to 1% of enzyme protein by weightof the composition, more preferably at a level of from 0.001% to 0.5% ofenzyme protein by weight of the composition, even more preferably at alevel of from 0.01% to 0.2% of enzyme protein by weight of thecomposition.

Lipases. Any lipase suitable for use in alkaline solutions can be used.Suitable lipases include those of bacterial or fungal origin. Chemicallyor genetically modified mutants are included.

Examples of useful lipases include a Humicola lanuginosa lipase, e.g.,as described in EP 258 068 and EP 305 216, a Rhizomucor miehei lipase,e.g., as described in EP 238 023, a Candida lipase, such as a C.antarctica lipase, e.g., the C. antarctica lipase A or B described in EP214 761, a Pseudomonas lipase such as a P. alcaligenes and P.pseudoalcaligenes lipase, e.g., as described in EP 218 272, a P. cepacialipase, e.g., as described in EP 331 376, a P. stutzeri lipase, e.g., asdisclosed in GB 1,372,034, a P. fluorescens lipase, a Bacillus lipase,e.g., a B. subtilis lipase (Dartois et al., 1993, Biochemica etBiophysica acta 1131, 253-260), a B. stearothermophilus lipase (JP64/744992) and a B. pumilus lipase (WO 91/16422).

Furthermore, a number of cloned lipases may be useful, including thePenicillium camembertii lipase described by Yamaguchi et al., 1991, Gene103, 61-67), the Geotricum candidum lipase (Schimada, Y. et al., 1989,J. Biochem., 106, 383-388), and various Rhizopus lipases such as a R.delemar lipase (Hass, M. J et al., 1991, Gene 109, 117-113), a R. niveuslipase (Kugimiya et al., 1992, Biosci. Biotech. Biochem. 56, 716-719)and an R. oryzae lipase.

Other types of lipolytic enzymes such as cutinases may also be useful,e.g., a cutinase derived from Pseudomonas mendocina as described in WO88/09367, or a cutinase derived from Fusarium solani pisi (e.g.,described in WO 90/09446).

Especially suitable lipases are lipases such as M1 Lipase™, Luma fast™and Lipomax™ (Genencor), Lipolase™ and Lipolase Ultra™ (Novo NordiskA/S), and Lipase P “Amano” (Amano Pharmaceutical Co. Ltd.).

The lipases are normally incorporated in the detergent composition at alevel of from 0.00001% to 2% of enzyme protein by weight of thecomposition, preferably at a level of from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level of from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level of from 0.01% to 0.2% of enzyme protein by weightof the composition.

Amylases: Any amylase (alpha and/or beta) suitable for use in alkalinesolutions can be used. Suitable amylases include those of bacterial orfungal origin. Chemically or genetically modified mutants are included.Amylases include, for example, a-amylases obtained from a special strainof B. licheniformis, described in more detail in GB 1,296,839.Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ andBAN™ (available from Novo Nordisk A/S) and Rapidase™ and Maxamyl P™(available from Genencor).

The amylases are normally incorporated in the detergent composition at alevel of from 0.00001% to 2% of enzyme protein by weight of thecomposition, preferably at a level of from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level of from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level of from 0.01% to 0.2% of enzyme protein by weightof the composition.

Cellulases: Any cellulase suitable for use in alkaline solutions can beused. Suitable cellulases include those of bacteria or fungal origin.Chemically or genetically modified mutants are included. Suitablecellulases are disclosed in U.S. Pat. No. 4,435,307, which disclosesfungal cellulases produced from Humicola insolens. Especially suitablecellulases are the cellulases having color care benefits. Examples ofsuch cellulases are cellulases described in European patent applicationNo. 0 495 257 and the endoglucanase of the present invention.

Commercially available cellulases include Celluzyme™ produced by astrain of Humicola insolens (Novo Nordisk A/S) and KAC˜500(B)™ (KaoCorporation).

Cellulases are normally incorporated in the detergent composition at alevel of from 0.00001% to 2% of enzyme protein by weight of thecomposition, preferably at a level of from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level of from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level of from 0.01% to 0.2% of enzyme protein by weightof the composition.

Peroxidases/Oxidases: Peroxidase enzymes are used in combination withhydrogen peroxide or a source thereof (e.g., a percarbonate, perborateor persulfate). Oxidase enzymes are used in combination with oxygen.Both types of enzymes are used for “solution bleaching”, i.e., toprevent transfer of a textile dye from a dyed fabric to another fabricwhen said fabrics are washed together in a wash liquor, preferablytogether with an enhancing agent as described in, e.g., WO 94/12621 andWO 95/01426. Suitable peroxidases/oxidases include those of plant,bacterial or fungal origin. Chemically or genetically modified mutantsare included.

Peroxidase and/or oxidase enzymes are normally incorporated in thedetergent composition at a level of from 0.00001% to 2% of enzymeprotein by weight of the composition, preferably at a level of from0.0001% to 1% of enzyme protein by weight of the composition, morepreferably at a level of from 0.001% to 0.5% of enzyme protein by weightof the composition, even more preferably at a level of from 0.01% to0.2% of enzyme protein by weight of the composition.

Mixtures of the above mentioned enzymes are encompassed herein, inparticular a mixture of a protease, an amylase, a lipase and/or acellulase.

The enzyme of the invention, or any other enzyme incorporated in thedetergent composition, is normally incorporated in the detergentcomposition at a level from 0.00001% to 2% of enzyme protein by weightof the composition, preferably at a level from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level from 0.01% to 0.2% of enzyme protein by weight ofthe composition.

Bleaching Agents

Additional optional detergent ingredients that can be included in thedetergent compositions of the present invention include bleaching agentssuch as PB1, PB4 and percarbonate with a particle size of 400-800microns. These bleaching agent components can include one or more oxygenbleaching agents and, depending upon the bleaching agent chosen, one ormore bleach activators. When present oxygen bleaching compounds willtypically be present at levels of from about 1% to about 25%. Ingeneral, bleaching compounds are optional added components in non-liquidformulations, e.g., granular detergents.

The bleaching agent component for use herein can be any of the bleachingagents useful for detergent compositions including oxygen bleaches aswell as others known in the art.

The bleaching agent suitable for the present invention can be anactivated or non-activated bleaching agent.

One category of oxygen bleaching agent that can be used encompassespercarboxylic acid bleaching agents and salts thereof, Suitable examplesof this class of agents include magnesium monoperoxyphthalatehexahydrate, the magnesium salt of meta-chloro perbenzoic acid,4-nonylamino-4-oxoperoxybutyric acid and diperoxydodecanedioic acid.Such bleaching agents are disclosed in U.S. Pat. No. 4,483,781, U.S.Pat. No. 740,446, EP 0 133 354 and U.S. Pat. No. 4,412,934. Highlypreferred bleaching agents also include 6-nonylamino-6-oxoperoxycaproicacid as described in U.S. Pat. No. 4,634,551.

Another category of bleaching agents that can be used encompasses thehalogen bleaching agents. Examples of hypohalite bleaching agents, forexample, include trichloro isocyanuric acid and the sodium and potassiumdichloroisocyanurates and N-chloro and N-bromo alkane sulphonamides.Such materials are normally added at 0.5-10% by weight of the finishedproduct, preferably 1-5% by weight.

The hydrogen peroxide releasing agents can be used in combination withbleach activators such as tetra-acetylethylenediamine (TAED),nonanoyloxybenzenesulfonate (NOBS, described in U.S. Pat. No.4,412,934), 3,5-trimethyl-hexsanoloxybenzenesulfonate (ISONOBS,described in EP 120 591) or pentaacetylglucose (PAG), which areperhydrolyzed to form a peracid as the active bleaching species, leadingto improved bleaching effect. In addition, very suitable are the bleachactivators C8 (6-octanamido-caproyl) oxybenzene-sulfonate, C9(6-nonanamido caproyl) oxybenzenesulfonate and C10 (6-decanamidocaproyl) oxybenzenesulfonate or mixtures thereof. Also suitableactivators are acylated citrate esters such as disclosed in EuropeanPatent Application No. 91870207.7.

Useful bleaching agents, including peroxyacids and bleaching systemscomprising bleach activators and peroxygen bleaching compounds for usein cleaning compositions according to the invention are described inapplication U.S. Ser. No. 08/136,626.

The hydrogen peroxide may also be present by adding an enzymatic system(i.e., an enzyme and a substrate therefore) which is capable ofgeneration of hydrogen peroxide at the beginning or during the washingand/or rinsing process. Such enzymatic systems are disclosed in EuropeanPatent Application EP 0 537 381.

Bleaching agents other than oxygen bleaching agents are also known inthe art and can be utilized herein. One type of non-oxygen bleachingagent of particular interest includes photoactivated bleaching agentssuch as the sulfonated zinc and/or aluminium phthalocyanines. Thesematerials can be deposited upon the substrate during the washingprocess. Upon irradiation with light, in the presence of oxygen, such asby hanging clothes out to dry in the daylight, the sulfonated zincphthalocyanine is activated and, consequently, the substrate isbleached. Preferred zinc phthalocyanine and a photoactivated bleachingprocess are described in U.S. Pat. No. 4,033,718. Typically, detergentcomposition will contain about 0.025% to about 1.25%, by weight, ofsulfonated zinc phthalocyanine.

Bleaching agents may also comprise a manganese catalyst. The manganesecatalyst may, e.g., be one of the compounds described in “Efficientmanganese catalysts for low-temperature bleaching”, Nature, 1994, Vol.369, pp. 637-639.

Suds Suppressors

Another optional ingredient is a suds suppressor, exemplified bysilicones, and silica-silicone mixtures. Silicones can generally berepresented by alkylated polysiloxane materials, while silica isnormally used in finely divided forms exemplified by silica aerogels andxerogels and hydrophobic silicas of various types. Theses materials canbe incorporated as particulates, in which the suds suppressor isadvantageously releasably incorporated in a water-soluble orwater-dispersible, substantially non surface-active detergentimpermeable carrier. Alternatively the suds suppressor can be dissolvedor dispersed in a liquid carrier and applied by spraying on to one ormore of the other components.

A preferred silicone suds controlling agent is disclosed in U.S. Pat.No. 3,933,672. Other particularly useful suds suppressors are theself-emulsifying silicone suds suppressors, described in German PatentApplication DTOS 2,646,126. An example of such a compound is DC-544,commercially available form Dow Corning, which is a siloxane-glycolcopolymer. Especially preferred suds controlling agent are the sudssuppressor system comprising a mixture of silicone oils and2-alkyl-alkanols. Suitable 2-alkyl-alkanols are 2-butyl-octanol whichare commercially available under the trade name Isofol 12 R.

Such suds suppressor system are described in European Patent ApplicationEP 0 593 841.

Especially preferred silicone suds controlling agents are described inEuropean Patent Application No. 92201649.8. Said compositions cancomprise a silicone/silica mixture in combination with fumed nonporoussilica such as Aerosil®.

The suds suppressors described above are normally employed at levels offrom 0.001% to 2% by weight of the composition, preferably from 0.01% to1% by weight.

Other Components

Other components used in detergent compositions may be employed such assoil-suspending agents, soil-releasing agents, optical brighteners,abrasives, bactericides, tarnish inhibitors, coloring agents, and/orencapsulated or nonencapsulated perfumes.

Especially suitable encapsulating materials are water soluble capsuleswhich consist of a matrix of polysaccharide and polyhydroxy compoundssuch as described in GB 1,464,616.

Other suitable water soluble encapsulating materials comprise dextrinsderived from ungelatinized starch acid esters of substituteddicarboxylic acids such as described in U.S. Pat. No. 3,455,838. Theseacid-ester dextrins are, preferably, prepared from such starches as waxymaize, waxy sorghum, sago, tapioca and potato. Suitable examples of saidencapsulation materials include N-Lok manufactured by National Starch.The N-Lok encapsulating material consists of a modified maize starch andglucose. The starch is modified by adding monofunctional substitutedgroups such as octenyl succinic acid anhydride.

Antiredeposition and soil suspension agents suitable herein includecellulose derivatives such as methylcellulose, carboxymethylcellculoseand hydroxyethylcellulose, and homo- or co-polymeric polycarboxylicacids or their salts. Polymers of this type include the polyacrylatesand maleic anhydride-acrylic acid copolymers previously mentioned asbuilders, as well as copolymers of maleic anhydride with ethylene,methylvinyl ether or methacrylic acid, the maleic anhydride constitutingat least 20 mole percent of the copolymer. These materials are normallyused at levels of from 0.5% to 10% by weight, more preferably form 0.75%to 8%, most preferably from 1% to 6% by weight of the composition.

Preferred optical brighteners are anionic in character, examples ofwhich are disodium4,4′-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2:2′disulphonate, disodium4,4′-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino-stilbene-2:2′-disulphonate,disodium4,4′-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2:2′-disulphonate,monosodium 4′,4″-bis-(2,4-dianilino-s-tri-azin-6ylamino)stilbene-2-sulphonate, disodium4,4′-bis-(2-anilino-4-(N-methyl-N-2-hydroxyethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate,disodium4,4′-bis-(4-phenyl-2,1,3-triazol-2-yl)-stilbene-2,2′-disulphonate,disodium4,4′-bis(2-anilino-4-(1-methyl-2-hydroxyethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate,sodium 2(stilbyl-4″-(naphtho-1′,2′:4,5)-1,2,3-triazole-2″-sulphonate and4,4′-bis(2-sulphostyryl)biphenyl.

Other useful polymeric materials are the polyethylene glycols,particularly those of molecular weight 1000-10000, more particularly2000 to 8000 and most preferably about 4000. These are used at levels offrom 0.20% to 5% more preferably from 0.25% to 2.5% by weight. Thesepolymers and the previously mentioned homo- or co-polymericpoly-carboxylate salts are valuable for improving whiteness maintenance,fabric ash deposition, and cleaning performance on clay, proteinaceousand oxidizable soils in the presence of transition metal impurities.

Soil release agents useful in compositions of the present invention areconventionally copolymers or terpolymers of terephthalic acid withethylene glycol and/or propylene glycol units in various arrangements.Examples of such polymers are disclosed in U.S. Pat. Nos. 4,116,885 and4,711,730 and EP 0 272 033. A particular preferred polymer in accordancewith EP 0 272 033 has the formula:

(CH₃(PEG)43)0.75(POH)0.25[T-PO)2.8(T-PEG)0.4]T(POH)0.25((PEG)43CH₃)0.75where PEG is —(OC₂H₄)0-, PO is (OC₃H₆O) and T is (pOOC₆H₄CO).

Also very useful are modified polyesters as random copolymers ofdimethyl terephthalate, dimethyl sulfoisophthalate, ethylene glycol and1,2-propanediol, the end groups consisting primarily of sulphobenzoateand secondarily of mono esters of ethylene glycol and/or1,2-propanediol. The target is to obtain a polymer capped at both end bysulphobenzoate groups, “primarily”, in the present context most of saidcopolymers herein will be endcapped by sulphobenzoate groups. However,some copolymers will be less than fully capped, and therefore their endgroups may consist of monoester of ethylene glycol and/or1,2-propanediol, thereof consist “secondarily” of such species.

The selected polyesters herein contain about 46% by weight of dimethylterephthalic acid, about 16% by weight of 1,2-propanediol, about 10% byweight ethylene glycol, about 13% by weight of dimethyl sulfobenzoicacid and about 15% by weight of sulfoisophthalic acid, and have amolecular weight of about 3.000. The polyesters and their method ofpreparation are described in detail in EP 311 342.

Softening Agents

Fabric softening agents can also be incorporated into laundry detergentcompositions in accordance with the present invention. These agents maybe inorganic or organic in type. Inorganic softening agents areexemplified by the smectite clays disclosed in GB-A-1 400 898 and inU.S. Pat. No. 5,019,292. Organic fabric softening agents include thewater insoluble tertiary amines as disclosed in GB-A-1 514 276 and EP 0011 340 and their combination with mono C12-C14 quaternary ammoniumsalts are disclosed in EP-B-0 026 528 and di-long-chain amides asdisclosed in EP 0 242 919. Other useful organic ingredients of fabricsoftening systems include high molecular weight polyethylene oxidematerials as disclosed in EP 0 299 575 and 0 313 146.

Levels of smectite clay are normally in the range from 5% to 15%, morepreferably from 8% to 12% by weight, with the material being added as adry mixed component to the remainder of the formulation. Organic fabricsoftening agents such as the water-insoluble tertiary amines or dilongchain amide materials are incorporated at levels of from 0.5% to 5% byweight, normally from 1% to 3% by weight whilst the high molecularweight polyethylene oxide materials and the water soluble cationicmaterials are added at levels of from 0.1% to 2%, normally from 0.15% to1.5% by weight. These materials are normally added to the spray driedportion of the composition, although in some instances it may be moreconvenient to add them as a dry mixed particulate, or spray them asmolten liquid on to other solid components of the composition.

Polymeric Dye-transfer Inhibiting Agents

The detergent compositions according to the present invention may alsocomprise from 0.001% to 10%, preferably from 0.01% to 2%, morepreferably form 0.05% to 1% by weight of polymeric dye-transferinhibiting agents. Said polymeric dye-transfer inhibiting agents arenormally incorporated into detergent compositions in order to inhibitthe transfer of dyes from colored fabrics onto fabrics washed therewith.These polymers have the ability of complexing or adsorbing the fugitivedyes washed out of dyed fabrics before the dyes have the opportunity tobecome attached to other articles in the wash.

Especially suitable polymeric dye-transfer inhibiting agents arepolyamine N-oxide polymers, copolymers of N-vinyl-pyrrolidone andN-vinylimidazole, polyvinylpyrrolidone polymers, polyvinyloxazolidonesand polyvinylimidazoles or mixtures thereof.

Addition of such polymers also enhances the performance of the enzymesaccording the invention.

The detergent composition according to the invention can be in liquid,paste, gels, bars or granular forms.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 (both to Novo Industri A/S) and mayoptionally be coated by methods known in the art. Examples of waxycoating materials are poly(ethylene oxide) products (polyethyleneglycol,PEG) with mean molecular weights of 1000 to 20000; ethoxylatednonylphenois having from 16 to 50 ethylene oxide units; ethoxylatedfatty alcohols in which the alcohol contains from 12 to 20 carbon atomsand in which there are 15 to 80 ethylene oxide units; fatty alcohols;fatty acids; and mono- and di- and triglycerides of fatty acids.Examples of film-forming coating materials suitable for application byfluid bed techniques are given in GB 1483591.

Granular compositions according to the present invention can also be in“compact form”, i.e., they may have a relatively higher density thanconventional granular detergents, i.e., form 550 to 950 g/l; in suchcase, the granular detergent compositions according to the presentinvention will contain a tower amount of “Inorganic filler salt”,compared to conventional granular detergents; typical filler salts arealkaline earth metal salts of sulphates and chlorides, typically sodiumsulphate; “Compact” detergent typically comprise not more than 10%filler salt. The liquid compositions according to the present inventioncan also be in “concentrated form”, in such case, the liquid detergentcompositions according to the present invention will contain a loweramount of water, compared to conventional liquid detergents. Typically,the water content of the concentrated liquid detergent is less than 30%,more preferably less than 20%, most preferably less than 10% by weightof the detergent compositions.

The compositions of the invention may for example, be formulated as handand machine laundry detergent compositions including laundry additivecompositions and compositions suitable for use in the pretreatment ofstained fabrics, rinse added fabric softener compositions, andcompositions for use in general household hard surface cleaningoperations and dishwashing operations.

The following examples are meant to exemplify compositions for thepresent invention, but are not necessarily meant to limit or otherwisedefine the scope of the invention.

In the detergent compositions, the abbreviated component identificationshave the following meanings:

LAS: Sodium linear C12 alkyl benzene sulphonateTAS: Sodium tallow alkyl sulphateXYAS: Sodium C1X-C1Y alkyl sulfateSS: Secondary soap surfactant of formula 2-butyl octanoic acid25EY: A C12-C15 predominantly linear primary alcohol condensed with anaverage of Y moles of ethylene oxide45EY: A C14-C15 predominantly linear primary alcohol condensed with anaverage of Y moles of ethylene oxideXYEZS: C1X-C1Y sodium alkyl sulfate condensed with an average of Z molesof ethylene oxide per moleNonionic: C13-C15 mixed ethoxylated/propoxylated fatty alcohol with anaverage degree of ethoxylation of 3.8 and an average degree ofpropoxylation of 4.5 sold under the tradename Plurafax LF404 by BASFGmbhCFAA: C12-C14 alkyl N-methyl glucamideTFAA: C16-C18 alkyl N-methyl glucamideSilicate: Amorphous Sodium Silicate (SiO₂:Na₂O ratio=2.0)NaSKS-6: Crystalline layered silicate of formula d-Na₂Si₂O₅Carbonate: Anhydrous sodium carbonatePhosphate: Sodium tripolyphosphateMA/AA: Copolymer of 1:4 maleic/acrylic acid, average molecular weightabout 80,000Polyacrylate: Polyacrylate homopolymer with an average molecular weightof 8,000 sold under the tradename PA30 by BASF GmbhZeolite A: Hydrated Sodium Aluminosilicate of formula Na₁₂(AlO₂SiO₂)₁₂.27H₂O having a primary particle size in the range from 1 to 10micrometersCitrate: Tri-sodium citrate dihydrate

Citric: Citric Acid

Perborate: Anhydrous sodium perborate monohydrate bleach, empiricalformula NaBO₂.H₂O₂PB4: Anhydrous sodium perborate tetrahydratePercarbonate: Anhydrous sodium percarbonate bleach of empirical formula2Na₂CO₃.3H₂O₂TAED: Tetraacetyl ethylene diamineCMC: Sodium carboxymethyl celluloseDETPMP: Diethylene triamine penta(methylene phosphonic acid), marketedby Monsanto under the Tradename Dequest 2060PVP: Polyvinylpyrrolidone polymerEDDS: Ethylenediamine-N, N′-disuccinic acid, [S,S] isomer in the form ofthe sodium saltSuds Suppressor: 25% paraffin wax Mpt 50° C., 17% hydrophobic silica,58% paraffin oilGranular Suds suppressor: 12% Silicone/silica, 18% stearyl alcohol, 70%starch in granular formSulphate: Anhydrous sodium sulphate

HMWPEO: High molecular weight polyethylene oxide

TAE 25: Tallow alcohol ethoxylate (25)

DETERGENT EXAMPLE I

A granular fabric cleaning composition in accordance with the inventionmay be prepared as follows:

Sodium linear C12 alkyl 6.5 benzene sulfonate Sodium sulfate 15.0Zeolite A 26.0 Sodium nitrilotriacetate 5.0 Enzyme of the invention 0.1PVP 0.5 TAED 3.0 Boric acid 4.0 Perborate 18.0 Phenol sulphonate 0.1Minors Up to 100

DETERGENT EXAMPLE II

A compact granular fabric cleaning composition (density 800 g/l) inaccord with the invention may be prepared as follows:

45AS 8.0 25E3S 2.0 25E5 3.0 25E3 3.0 TFAA 2.5 Zeolite A 17.0 NaSKS-612.0 Citric acid 3.0 Carbonate 7.0 MA/AA 5.0 CMC 0.4 Enzyme of theinvention 0.1 TAED 6.0 Percarbonate 22.0 EDDS 0.3 Granular sudssuppressor 3.5 water/minors Up to 100%

DETERGENT EXAMPLE III

Granular fabric cleaning compositions in accordance with the inventionwhich are especially useful in the laundering of coloured fabrics wereprepared as follows:

LAS 10.7  — TAS 2.4 — TFAA — 4.0 45AS 3.1 10.0  45E7 4.0 — 25E3S — 3.068E11 1.8 — 25E5 — 8.0 Citrate 15.0  7.0 Carbonate — 10   Citric acid2.5 3.0 Zeolite A 32.1  25.0  Na-SKS-6 — 9.0 MA/AA 5.0 5.0 DETPMP 0.20.8 Enzyme of the invention  0.10  0.05 Silicate 2.5 — Sulphate 5.2 3.0PVP 0.5 — Poly (4-vinylpyridine)-N- — 0.2 Oxide/copolymer of vinyl-imidazole and vinyl- pyrrolidone Perborate 1.0 — Phenol sulfonate 0.2 —Water/Minors Up to 100%

DETERGENT EXAMPLE IV

Granular fabric cleaning compositions in accordance with the inventionwhich provide “Softening through the wash” capability may be prepared asfollows:

45AS — 10.0 LAS 7.6 — 68AS 1.3 — 45E7 4.0 — 25E3 — 5.0Coco-alkyl-dimethyl hydroxy- 1.4 1.0 ethyl ammonium chloride Citrate 5.03.0 Na-SKS-6 — 11.0 Zeolite A 15.0 15.0 MA/AA 4.0 4.0 DETPMP 0.4 0.4Perborate 15.0 — Percarbonate — 15.0 TAED 5.0 5.0 Smectite clay 10.010.0 HMWPEO — 0.1 Enzyme of the invention 0.10 0.05 Silicate 3.0 5.0Carbonate 10.0 10.0 Granular suds suppressor 1.0 4.0 CMC 0.2 0.1Water/Minors Up to 100%

DETERGENT EXAMPLE V

Heavy duty liquid fabric cleaning compositions in accordance with theinvention may be prepared as follows:

I II LAS acid form — 25.0 Citric acid 5.0 2.0 25AS acid form 8.0 —25AE2S acid form 3.0 — 25AE7 8.0 — CFAA 5 — DETPMP 1.0 1.0 Fatty acid 8— Oleic acid — 1.0 Ethanol 4.0 6.0 Propanediol 2.0 6.0 Enzyme of theinvention 0.10 0.05 Coco-alkyl dimethyl — 3.0 hydroxy ethyl ammoniumchloride Smectite clay — 5.0 PVP 2.0 — Water/Minors Up to 100%

Textile Applications

In another embodiment, the present invention relates to use of theendoglucanase of the invention in the bio-polishing process,Bio-Polishing is a specific treatment of the yarn surface which improvesfabric quality with respect to handle and appearance without toss offabric wettability. The most important effects of Bio-Polishing can becharacterized by less fuzz and pilling, increased gloss/luster, improvedfabric handle, increased durable softness and altered water absorbency,Bio-Polishing usually takes place in the wet processing of themanufacture of knitted and woven fabrics. Wet processing comprises suchsteps as, e.g., desizing, scouring, bleaching, washing, dying/printingand finishing. During each of these steps, the fabric is more or lesssubjected to mechanical action. In general, after the textiles have beenknitted or woven, the fabric proceeds to a desizing stage, followed by ascouring stage, etc. Desizing is the act of removing size from textiles.Prior to weaving on mechanical looms, warp yarns are often coated withsize starch or starch derivatives in order to increase their tensilestrength. After weaving, the size coating must be removed before furtherprocessing the fabric in order to ensure a homogeneous and wash-proofresult. It is known that in order to achieve the effects ofBio-Polishing, a combination of cellulytic and mechanical action isrequired. It is also known that “super-softness” is achievable when thetreatment with a cellulase is combined with a conventional treatmentwith softening agents. It is contemplated that use of the endoglucanaseof the invention for bio-polishing of cellulosic fabrics isadvantageous, e.g., a more thorough polishing can be achieved.Bio-polishing may be obtained by applying the method described, e.g., inWO 93/20278.

Stone-Washing

It is known to provide a “stone-washed” look (localized abrasion of thecolor) in dyed fabric, especially in denim fabric or jeans, either bywashing the denim or jeans made from such fabric in the presence ofpumice stones to provide the desired localized lightening of the colorof the fabric or by treating the fabric enzymatically, in particularwith cellulytic enzymes. The treatment with an endoglucanase of thepresent invention may be carried out either alone such as disclosed inU.S. Pat. No. 4,832,864, together with a smaller amount of pumice thanrequired in the traditional process, or together with perlite such asdisclosed in WO 95/09225.

Pulp and Paper Applications

In the papermaking pulp industry, the endoglucanase of the presentinvention may be applied advantageously, e.g., as follows:

-   -   For debarking: pretreatment with the endoglucanase may degrade        the cambium layer prior to debarking in mechanical drums        resulting in advantageous energy savings.    -   For defibration: treatment of a material containing cellulosic        fibers with the endoglucanase prior to refining or beating may        result in reduction of the energy consumption due to the        hydrolyzing effect of the cellulase on the interfiber surfaces.        Use of the endoglucanase may result in improved energy savings        as compared to the use of known enzymes, since it is believed        that the enzyme composition of the invention may possess a        higher ability to penetrate fiber walls.    -   For fiber modification, i.e., improvement of fiber properties        where partial hydrolysis across the fiber wall is needed which        requires deeper penetrating enzymes (e.g., in order to make        coarse fibers more flexible). Deep treatment of fibers has so        far not been possible for high yield pulps, e.g., mechanical        pulps or mixtures of recycled pulps. This has been ascribed to        the nature of the fiber wall structure that prevents the passage        of enzyme molecules due to physical restriction of the pore        matrix of the fiber wall. It is contemplated that the present        endoglucanase is capable of penetrating into the fiber wall.    -   For drainage improvement. The drainability of papermaking pulps        may be improved by treatment of the pulp with hydrolysing        enzymes, e.g., cellulases. Use of the present endoglucanase may        be more effective, e.g., result in a higher degree of loosening        bundles of strongly hydrated micro-fibrils in the fines fraction        (consisting of fiber debris) that limits the rate of drainage by        blocking hollow spaces between fibers and in the wire mesh of        the paper machine. The Canadian standard freeness (CSF)        increases and the Schopper-Riegler drainage index decreases when        pulp in subjected to cellulase treatment, see, e.g., U.S. Pat.        No. 4,923,565; TAPPI T227, SCAN C19:65.ence.    -   For inter fiber bonding. Hydrolytic enzymes are applied in the        manufacture of papermaking pulps for improving the inter fiber        bonding. The enzymes rinse the fiber surfaces for impurities,        e.g., cellulosic debris, thus enhancing the area of exposed        cellulose with attachment to the fiber wall, thus improving the        fiber-to-fiber hydrogen binding capacity. This process is also        referred to as dehornification. Paper and board produced with a        cellulase containing enzyme preparation may have an improved        strength or a reduced grammage, a smoother surface and an        improved printability.    -   For enzymatic deinking. Partial hydrolysis of recycled paper        during or upon pulping by use of hydrolysing enzymes such as        cellulases are known to facilitate the removal and agglomeration        of ink particles. Use of the present endoglucanse may give a        more effective loosening of ink from the surface structure due        to a better penetration of the enzyme molecules into the        fibrillar matrix of the fiber wall, thus softening the surface        whereby ink particles are effectively loosened. The        agglomeration of loosened ink particles are also improved, due        to a more efficient hydrolysis of cellulosic fragments found        attached to ink particles originating from the fibers.

The treatment of lignocellulosic pulp may, e.g., be performed asdescribed in WO 91/14819, WO 91/14822, WO 92/17573 and WO 92/18688.

Degradation of Plant Material

In yet another embodiment, the present invention relates to use of theendoglucanase and/or enzyme preparation according to the invention fordegradation of plant material, e.g., cell walls.

It is contemplated that the novel endoglucanase and/or enzymepreparation of the invention is useful in the preparation of wine, fruitor vegetable juice in order to increase yield. Endoglucanases accordingto the invention may also be applied for enzymatic hydrolysis of variousplant cell-wall derived materials or waste materials, e.g., agriculturalresidues such as wheat-straw, corn cobs, whole corn plants, nut shells,grass, vegetable hulls, bean hulls, spent grains, sugar beet pulp, andthe like. The plant material may be degraded in order to improvedifferent kinds of processing, facilitate purification or extraction ofother components like purification of beta-glucan or beta-glucanoligomers from cereals, improve the feed value, decrease the waterbinding capacity, improve the degradability in waste water plants,improve the conversion of, e.g., grass and corn to ensilage, etc.

EXAMPLES

The invention is further illustrated in the following examples which arenot intended to be in any way limiting to the scope of the invention asclaimed.

Materials and Methods Cellulolytic Activity

The cellulase variants of the invention show improved performance. Someof the variants may show improved performance with respect to increasedcatalytic activity.

In the context of this invention, cellulase activity can be expressed inS-CEVU. Cellulolytic enzymes hydrolyse CMC, thereby increasing theviscosity of the incubation mixture. The resulting reduction inviscosity may be determined by a vibration viscosimeter (e.g., MIVI 3000from Sofraser, France).

Determination of the cellulolytic activity, measured in terms of S-CEVU,may be determined according to the following analysis method (assay):The S-CEVU assay quantifies the amount of catalytic activity present inthe sample by measuring the ability of the sample to reduce theviscosity of a solution of carboxy-methylcellulose (CMC). The assay iscarried out at 40° C.; pH 7.5; 0.1 M phosphate buffer; time 30 min;using a relative enzyme standard for reducing the viscosity of the CMC(carboxymethylcellulose Hercules 7 LFD) substrate; enzyme concentrationapprox, 0.15 S—CEVU/ml. The arch standard is defined to 8200 S-CEVU/g.

Example 1 Preparation of Cellulase Variants

Based on the disclosed sequence alignment (Table 1) and computermodeling method, position 119 was identified as a particular point ofinterest for making cellulase variants. Position 119 (cellulasenumbering) is located within 3 Å from the substrate. In position 119 thewild-type Humicola insolens cellulase holds a histidine residue (H)whereas the wild-type Thielavia terrestris cellulase holds a glutamineresidue (Q).

In this experiment, histidine was substituted for glutamine in theThielavia terrestris cellulase (thereby obtaining the cellulase variantThielavia terrestris/Q119H). The variant obtained was tested forspecific activity.

All Humicola insolens variants are, unless otherwise stated, constructedby application of the Chameleon™ Double-stranded, site-directedMutagenesis kit, from Stratagene. The following syntheticoligo-nucleotides were used as selection primers:

S/M GAATGACTTGGTTGACGCGTCACCAGTCAC, (SEQ ID NO: 12) or M/SGAATGACTTGGTTGAGTACTCACCAGTCAC. (SEQ ID NO: 13)

S/M replaces the ScaI site in the beta-lactamase gene of the plasmidwith a MIuI site and M/S does the reverse. The latter is used tointroduce secondary mutations in variants generated by the firstselection primer.

For construction of Thielavia terrestis cellulase variants, theThielavia terrestis EG V cellulase cDNA obtainable from the plasmiddeposited as DSM 10811 was used. DSM 10811 was deposited at the DeutscheSammlung von Mikroorganismen und Zellkulturen on 30 Jun. 1995 accordingto the Budapest Treaty. The plasmid was digested with the restrictionendonucleases BamHI and NotI The 4153 bp vector part and the 1211 bpBamHI-NotI fragment were isolated. Equal portions of the 1211 bpfragment were digested with respectively HgiAl and EcoRV and the 487 bpBamHI-HgiAl and 690 bp EcoRV-NotI fragments were isolated.

These fragments and the vector part were ligated in the presence of 5fold molar excess of a synthetic DNA fragment, resulting from theannealing of two single stranded DNA oligomers:

(SEQ ID NO: 14) 18802: CACTGGCGGCGACCTGGGATCTAACCACTTCGAT (SEQ ID NO:15) 18803: ATCGAAGTGGTTAGATCCCAGGTCGCCGCCTGTGCTC

The ligation mixture was transformed into E. coli strain XL1, and fromthe resulting transformants Thielavia terrestris/Q119H was isolated andverified by DNA sequencing.

All the cellulase variants ware produced by cloning the gene andtransforming the gene into Aspergillus oryzae using a plasmid with thegene inserted between the fungal amylase promoter and the AMG terminatorfrom A. niger [Christensen, T. Wöldike, H. Boel, E., Mortensen, S. B.,Hjortshøj, K., Thim, L. and Hansen, M. T. (1988) Biotechnology 6:1419-1422].

The cellulases with a cellulose binding domain CBD were purified byexploiting their binding to Avicel. The cloned product was recoveredafter fermentation by separation of the extracellular fluid from theproduction organism. The cellulase was then highly purified by affinitychromatography using 150 gram of Avicel in a slurry with 20 mMsodiumphosphate pH 7.5. The Avicel slurry was mixed with the crudefermentation broth which in total contains about 1 gram of protein.After mixing at 4° C. for 20 min, the Avicel-bound enzyme is packed intoa column with a dimension of 50 times 200 mm about 400 ml total.

The column is washed with the 200 ml buffer, then washed with 0.5 M NaClin the same buffer until no more protein elutes, and washed with 500 mlbuffer (20 mM Tris pH 8.5). Finally the pure full length enzyme iseluted with 1% Triethylamine pH 11.8. The eluted enzyme solution isadjusted to pH 8 and concentrated using an Amicon cell unit with amembrane DOW GR61PP (polypropylene with a cut off of 20 KD) to 5 mgprotein per ml. The enzymes have all been purified yielding a singleband on SDS-PAGE.

Cellulases which natural lack CBD or the linker has been proteolyticcleaved or in which the CBD has been removed by introducing a stop codonafter the catalytic domain, can not be purified using Avicel. Theextracellular proteins are recovered free from the production organism.The core cellulases were purified free of Aspergillus proteins by cationexchange chromatography. The fermentation broth was adjusted to pH 3.5and filtered to remove the precipitating proteins. Then the proteinswere ultra filtrated (concentrated and washed with water) on a DOWGR81PP membrane with a cut off 6 KD until the conductivity of the eluateis below 1000 mS/cm. The sample was finally applied to an S-Sepharosecolumn equilibrated with a 20 mM citrate buffer pH 3.5.

The enzyme will bind to the S-Sepharose at this low pH and it is elutedas a single peak using a NaCl gradient from 0 to 500 mM. The eluted pureenzyme was concentrated on a Amicon cell with the DOW GR81PP membrane.All purified cellulases gave a single band in SDS-PAGE.

The specific activity data are summarized in the following table:

Enzyme/variant Specific activity [%] Humicola insolens 100 Thielaviaterrestris 35 Thielavia terrestris/Q119H 92

From this experiment it is seen that by introducing the mutation Q119Hinto the Thielavia terrestris cellulase, the specific activity if theresulting cellulase variants was increased to the level of that of thehomologous Humicola insolens cellulase.

Example 2 Thielavia terrestris Variant with Improved AlkalinePerformance Profile

In this experiment the Thielavia terrestris/Q119D was constructed asdescribed in example 1 but using the following construction: For easycassette swap and standard primer utilization, the CT1 encoding DNA wasfurnished with a C-terminal Xba1 site and subcloned into the pCaHj418vector as described below. PCT1 was used as template in a Pwo polymerasePCR, 94° C., 2′-3×(94° C., 30″-72° C., 1′)-25×(94° C., 30″-55° C.,30″-72° C., 1′)-72° C., 5′ applying the two primers

8939: CGACTTCAATGTCCAGTCGG (SEQ ID NO: 16) 25335:GCGCTCTAGAGGATTAAAGGCACTGC (SEQ ID NO: 17)

The resulting 718 bp PCR product was digested with Sal1 and Xba1 and the165 bp fragment was isolated. This fragment was ligated together withthe 833 bp BamH1-Sal1 fragment from pCT1-2 into the 4.1 kb Xba1-BamH1vector fragment of pCaHj418.

From this ligation pCT1418 was isolated from E. coli transformants.

PCT2 was constructed by the Chameleon™ Double-stranded, site-directedMutagenesis kit (from Stratagene) as described above with pCT1418 astemplate, the S/M primer as selection primer and the following mutagenicprimer:

(SEQ ID NO: 18) 109330: CGACCTGGGATCGAACGACTTCGATATCGCCATGC

A successfully mutated plasmid pCT2 was isolated, verified by DNAsequencing and transformed into Aspergillus oryzae strain JaL228.

The Thielavia terrestris cellulase and the Thielavia terrestris/Q119Dvariant was tested for activity towards PASC as described in example 9at pH 7.0 and pH 10.0.

The results are presented in the table below which shows the activity atpH10 compared to the activity at pH 7. This demonstrates that theThielavia terrestris/Q119D variant has relatively more alkaline activityas compared to the parent Thielavia terrestris.

Relative activity pH 10/pH 7 [%] Thielavia terrestris 27 Thielaviaterrestris/Q119D 62

Example 3

Construction of a Cellulase Hybrid Variant

The plasmid pCT3 embodies DNA encoding the Thielavia terrestrisendoglucanase core enzyme and followed by the linker COB of Humicolagrisea.

pCT3 was constructed by means of sequence overlap extension PCR,applying PWO polymerase.

From a cDNA clone of Humicola grisea a 415 bp fragment was generated bythe following primers:

(SEQ ID NO: 19) 109452: CGACTCCAGCTTCCCCGTCTTCACGCCCCC (SEQ ID NO: 20)107819: CGAGCTTCTAGATCTCGACTAGAGGCACTGGGAG

From pCT1418 (disclosed in example 2) an 876 bp PCR fragment wasgenerated by the following primers:

(SEQ ID NO: 21) 101621: GGATGCCATGCTTGGAGGATAGCAACC (SEQ ID NO: 22)107823: GGGGGCGTGAACAGGGGAAGCTGGAGTCG

For both reactions the following set up was used: 96° C., 1′-3×(94° C.,30″-50° C., 1′-72° C., 1′)-25×(94° C., 30″-61° C., 30″-72° C., 1′)-72°C., 7′.

The isolated PCR fragments were applied as template in an assembly PCRreaction with primers 101621 and 107819: 94° C., 1′-3×(94° C. 30″-70°C., 1′-72° C., 2′)-20×(94° C. 30″-61° C., 30″-72° C., 1.5′)-72° C., 7′.The resulting 1261 bp PCR product was isolated cut by restrictionenzymes BamH1 and Xba1 and the resulting 1172 bp DNA fragment wasisolated and ligated into the 4.1 kb vector fragment of BamH1-Xba1digested pCaHj418.

Correct clones were isolated and verified by DNA sequencing of plasmidsisolated from E. coli XL1 transformants resulting above ligationreaction.

cdNA Sequence of Humicola grisea (SEQ ID NO: 23):

CAAGAACCTCACACTCAATTTTATTCACGCTCATTTATTCTAAAACTTCAATATGCGCTCTGCTCCTATTTTCCGCACGGCCCTGGCGGCTGCGCTCCCCCTTGCCGCACTCGCCGCCGATGGCAAGTCGACCAGATACTGGGACTGCTGCAAGCCATCGTGCTCTTGGCCCGGAAAGGCACTCGTGAACCAGCCTGTCTTCACTTGCGACGCCAAATTCCAGCGCATCACCGACCCCAATACCAAGTCGGGCTGCGATGGCGGCTCGGCCTTTTCGTGTGCTGACCAGACCCCCTGGGCTCTGAACGACGATGTCGCCTATGGCTTCGCTGCCACGGCTATTTCGGGTGGATCGGAAGCCTCGTGGTGCTGCGCATGCTACGCTCTTACTTTCACCTCGGGCCCTGTGGCCGGCAAGACCATGGTCGTCCAGTCGACCAACACCGGCGGCGATCTCGGCAGCAACCATTTCGACCTCCAGATTCCAGGCGGCGGTGTCGGCATCTTTGATGGGTGCACCCCCCAGTTCGGAGGTCTCGCTGGCGAACGCTACGGTGGCATCTCAGACCGCAGCTCCTGCGACTCGTTCCCTGCGGCGCTCAAGCCCGGCTGCCTCTGGCGCTTCGATTGGTTCAAGAACGCCGACAACCCGACCTTTACCTTCAAGCAGGTGCAGTGCCCCGCCGAGCTTGTTGCCAGGACCGGCTGCAAGCGCGAGGATGACGGCAACTTCCCCGTCTTCACGCCCCGCCGCGGGTAGCAACACCGGCGGTAGCCAGTCGAGCTCCACTATCGCTTCCAGCTCGACCTCCAAGGCTCAGACTTCGGCCGCCAGCTCCACCTCCAAGGCTGTCGTGACTCCCGTCTCCAGCTCCACCTCGAAGGCCGCTGAGGTCCCCAAATCCAGCTCGACCTCCAAGGCTGCCGAGGTCGCCAAGCCCAGCTCAACTTCGACCTCGACCTCGACCTCGACCAAGGTCAGCTGCTCTGCGACCGGTGGCTCCTGCGTCGCTCAGAAGTGGGCGCAGTGCGGCGGCAATGGCTTCACCGGCTGCACGTCGTGCGTCAGCGGCACCACCTGCCAGAAGCAAAATGACTGGTACTCCCAGTGCCTCTAAGTCGTTTGTAGTAGCAGTTTGAAGGATGTCAGGGATGAGGGAGGGAGGAGTGGGGGAAAAGTACGCCGCAGTTTTTTGGTAGACTTACTGTATTGTTGAGTAATTACCCATTCGCTTCTTGTACGAAAAAAA AAAAAAAAAAAAA

Example 4 Construction of Variants of a Hybrid Cellulase

The plasmid pPsF45 embodies DNA encoding the Pseudomonas cellolyticaendoglucanase core enzyme headed by the H. insolens EGV endoglucanasesignal peptide and followed by the linker CBD of same enzyme.

Two variants of this hybrid enzyme were constructed by means of theabove-mentioned Stratagene Chameleon® kit:

PsF45/H15S and PsF45/Q119H (cellulase numbering) by application of thefollowing mutagenic primers

PsF45/H155: (SEQ ID NO: 24) GCTGCAAGCCGTCCTGTGGCTGGAGCGCTAACGTGCCCGCGPsF45/Q119H: (SEQ ID NO: 25) CGATGTTTCCGGAGGCCACTTTGACATTCTGGTTCC

Deviations from template sequence are indicated in bold type.

The selection primer was converting the unike Sca1 site in the lactamasegene of the plasmid to a Mlu1 site:

GAATGACTTGGTTGACGCGTCACCAGTCAC (SEQ ID NO: 26)

The two variants were verified by DNA sequencing and one correct versionof each variant was identified.

The two plasmids emharboring the variant sequences pPsF45H15S andpPsF45Q119H were used to transform A. oryzae strain JaL142 together withthe AMDS selection plasmid pToC202. From the resulting transformantsLaC2829 and LaC 2830 were isolated after 3 reisolation steps via spores.

Example 5 Removal of Disulfide Bridges

Disulfide bridges are known to stabilize protein structures. The removalof disulfide bridges in a cellulase will destabilizes the enzyme(thermostability) while retaining significant activity. This can beuseful in applications where a fast inactivation of the enzyme ispreferred, e.g., in denim or textile applications or for low temperatureprocesses.

In this example Humicola insolens EGV cellulase and five variants ofHumicola insolens cellulase were constructed mutating either one or bothresidues involved in a disulfide bridge. The specific activity wasmeasured as disclosed under Materials and Methods. The meltingtemperature of the enzymes was measured using Differential ScanningCalometry, DSC. DSC was done at neutral pH (7.0) using a MicroCalc Inc.MC calorimeter with a constant scan rate and raising the temperaturefrom 20° C. to 90° C. at a rate of 90° C. per hour.

The results are presented in the table below which shows that removal ofa disulfide bridge leads to a variant with a significantly lower meltingtemperature but retaining significant activity.

Melting Specific activity [%] temp. [° C.] Humicola insolens 100 81Humicola insolens/C12G, C47M 15 63.7 Humicola insolens/C12M, C47G 5364.3 Humicola insolens/C47G 48 57.3 Humicola insolens/C87M, C199G 7563.4 Humicola insolens/C16M, C86G 103 59.2

Example 6 Mutation of Conserved Residues in the Binding Cleft<5 Å fromsubstrate

When comparing the positions within a distance of 5 Å from the substrateto the sequence alignment in Table 1 the type of amino acid residue atthese positions are conserved in the aligned cellulases for thefollowing positions: 6, 7, 8, 9, 10, 11, 12, 18, 45, 112, 114, 121, 127,128, 130, 132, 147, 148, and 149. Conserved residues are normallythought to be extremely important for the activity, but the inventorshave found that a certain variability is allowed while maintainingsignificant activity. Only the two residues D10 and D121 (cellulasenumbering) are necessary to maintain reasonable activity.

Variants of the Humicola insolens EGV cellulase were prepared and thespecific activity was measured as disclosed in Materials and Methods.

The type of mutations and the variants specific activity are summarizedin the following table:

Variant Specific activity [%] Humicola insolens 100 Humicolainsolens/T6S 34 Humicola insolens/R7I 33 Humicola insolens/R7W 29Humicola insolens/Y8F 67 Humicola insolens/W9F 83 Humicolainsolens/C12M, C47G 53 Humicola insolens/W18Y 49 Humicola insolens/W18F53 Humicola insolens/S45T 85 Humicola insolens/S45N 85 Humicolainsolens/D114N 6 Humicola insolens/F132D 11 Humicola insolens/Y147D 34Humicola insolens/Y147C 30 Humicola insolens/Y147W 74 Humicolainsolens/Y147V 33 Humicola insolens/Y147R 45 Humicola insolens/Y147G 34Humicola insolens/Y147Q 41 Humicola insolens/Y147N 53 Humicolainsolens/Y147K 45 Humicola insolens/Y147H 75 Humicola insolens/Y147F 57Humicola insolens/Y147S 55

From this experiment it is seen that mutating conserved residues in thebinding cleft can be performed while retaining significant activity ofthe cellulose variant.

Example 7 Mutation of Non-Conserved Residues in the Binding Cleft<5 Åfrom the Substrate

Based on the sequence alignment in Table 1 and the disclosed computermodeling method the following residues located within a distance of 5 Åfrom the substrate and not being conserved amongst the aligned sequencesin were identified as points of interest for making cellulase variants.

In this experiment non-conserved residues located no more than 5 Å fromthe substrate were modified in the Humicola insolens EGV cellulase andthe specific activity was measured as described under Materials andMethods.

The type of mutations and the variants specific activity are summarizedin the following table:

Specific activity [%] Humicola insolens 100 Humicola insolens/R4H 73Humicola insolens/R4Q 70 Humicola insolens/K13L 37 Humicolainsolens/K13R 100 Humicola insolens/K13Q 38 Humicola insolens/P14A 99Humicola insolens/P14T 71 Humicola insolens/S15T 18 Humicolainsolens/S15H 10 Humicola insolens/C16M, C86G 103 Humicola insolens/A19P51 Humicola insolens/A19T 84 Humicola insolens/A19G 78 Humicolainsolens/A19S 89 Humicola insolens/K20G 91 Humicola insolens/D42Y 102Humicola insolens/D42W 103 Humicola insolens/C47G 48 Humicolainsolens/E48D 93 Humicola insolens/E48Q 71 Humicola insolens/E48D, P49*88 Humicola insolens/E48N, P49* 79 Humicola insolens/S110N 94 Humicolainsolens/L115I 18 Humicola insolens/G116D 71 Humicola insolens/H119R 15Humicola insolens/H119Q 39 Humicola insolens/H119F 11 Humicolainsolens/N123A 61 Humicola insolens/N123M 80 Humicola insolens/N123Q 76Humicola insolens/N123Y 8 Humicola insolens/N123D 86 Humicolainsolens/V129L 72 Humicola insolens/D133N 102 Humicola insolens/D178N 81

From this experiment it is seen that most of the non-conserved residuesin the binding cleft can be mutated while retaining all or most of theactivity of the cellulase.

Example 8 Resistance to Anionic Surfactants in Detergent

A. Variants of the present invention may show improved performance withrespect to an altered sensitivity towards anionic tensides. Anionictensides are products frequently incorporated into detergentcompositions. Unfolding of cellulases tested so far, is accompanied by adecay in the intrinsic fluorescence of the proteins. The intrinsicfluorescence derives from Trp side chains (and to a smaller extent Tyrside chains) and is sensitive to the hydrophobicity of the side chainenvironment. Unfolding leads to a more hydrophilic environment as theside-chains become more exposed to solvent, and this quenchesfluorescence.

Fluorescence is followed on a Perkin/Elmer™ LS50 luminescencespectrometer. In practice, the greatest change in fluorescence onunfolding is obtained by excitation at 280 nm and emission at 345 nm.Slit widths (which regulate the magnitude of the signal) are usually 5nm for both emission and excitation at a protein concentration of 5micrograms/ml. Fluorescence is measured in 2-ml quartz cuvettesthermostatted with a circulating water bath and stirred with a smallmagnet. The magnet-stirrer is built into the spectrometer.

Unfolding can be followed in real time using the available software.Rapid unfolding (going to completion within less than 5-10 minutes) ismonitored in the TimeDrive option, in which the fluorescence is measuredevery few (2-5) seconds. For slower unfolding, four cuvettes can bemeasured at a time in the cuvette-holder using the Wavelength Programoption, in which the fluorescence of each cuvette is measured every 30seconds. In all cases, unfolding is initiated by adding a small volume(typically 50 microliters) of concentrated enzyme solution to thethermostatted cuvette solution where mixing is complete within a fewseconds due to the rapid rotation of the magnet.

Data are measured in the software program GraphPad Prism. Unfolding fitsin all cases to a single-exponential function from which a singlehalf-time of unfolding (or unfolding rate constant) can be obtained.

Typical unfolding conditions are:

a. 10 mM CAPS pH 10, 1000 ppm LAS, 40° C. b. 10 mM HEPES pH 10, 200 ppmLAS, 25° C.

In both cases, the protein concentration is 5-10 micrograms/ml (theprotein concentration is not crucial, as LAS is in excess). Under theseconditions, the unfolding of Humicola insolens cellulase can be comparedwith other enzymes (Table 1). This enables us to draw up the followingranking order for stability against anionic tenside:

Thielavia terrestris/Q119H≈Thielavia terrestris>>Humicolainsolens≈Humicola insolens/H119Q.

t½ pH 7 (s) t½ pH 10 (s) (200 ppm LAS, Cellulase (1000 ppm LAS, 40° C.)25° C.) Humicola insolens 48 28 Humicola insolens/H119Q 63 9a Thielaviaterrestris 970 690 Thielavia terrestris/Q119H 1100 550a Unfolding is double-exponential. The t½ of the slower phase is approx.120 sec.

B. The alteration of the surface electrostatics of an enzyme willinfluence the sensibility towards anionic tensides such as LAS (linearalkylbenzenesulfonate). Especially variants where positive chargedresidues have been removed and/or negatively charged residues have beenintroduced will increase the resistance towards LAS, whereas theopposite, i.e., the introduction of positively charged residues and/orthe removal of negatively charged residues will lower the resistancetowards LAS. The residues Arg (R), Lys (K) and His (H) are viewed aspositively or potentially positively charged residue and the residuesAsp (D), Glu (E) and Cys (C) if not included in a disulphide bridge areviewed as negatively or potentially negatively charged residues.Positions already containing one of these residues are the primarytarget for mutagenesis, secondary targets are positions which have oneof these residues on an equivalent position in another cellulase, andthird target are any surface exposed residue. In this experiment wildtype Humicola insolens cellulase are being compared to Humicola insolenscellulose variants belonging to all three of the above groups, comparingthe stability towards LAS in detergent.

Cellulase resistance to anionic surfactants was measured as activity onPASC (phosphoric acid swollen cellulose) in the presence of anionicsurfactant vs. activity on PASC in the absence of anionic surfactant.

The reaction medium contained 5.0 g/l of a commercial regular powderdetergent from the detergent manufacturer NOPA Denmark. The detergentwas formulated without surfactants for this experiment and pH adjustedto pH 7.0. Further the reaction medium included 0.5 g/l PASC and waswith or without 1 g/l LAS (linear alkylbenzenesulphonate), which is ananionic surfactant, and the reaction proceeded at the temperature 30° C.for 30 minutes. Cellulase was dosed at 0.20 S-CEVU/I. After the 30minutes of incubation the reaction was stopped with 2 N NaOH and theamount of reducing sugar ends determined through reduction ofp-hydroxybenzoic acid hydrazide. The decrease in absorption of reducedp-hydroxybenzoic acid hydrazide relates to the cellulase activity.

The type of mutation and the resistance towards LAS for variants withincreased LAS resistance is summarized in the following table:

Relative Variant LAS resistance [%] Humicola insolens 100 Humicolainsolens/R158E 341 Humicola insolens/Y8F, W62E, A162P, 179 Humicolainsolens/R158E, A162P 347 Humicola insolens/R158G 322 Humicolainsolens/S152D 161 Humicola insolens/R158E/R196E 319 Humicolainsolens/R158E, D161P, A162P 351 Humicola insolens/R4H, R158E, D161P,A162P 344 Humicola insolens/H119Q 148 Humicola insolens/Y8F, W62E,R252L, Y280F 131 Humicola insolens/R252L, Y280F 133 Humicolainsolens/W62E, A162P 130 Humicola insolens/W62E, A162P 129 Humicolainsolens/S117D 143 Humicola insolens/A57C, A162C 134 Humicolainsolens/N154D 149 Humicola insolens/R4H, D161P, A162P, R196E 134

From this table it is seen that mutations of residues resulting in theremoval of positively charged residue and/or the introduction of anegatively charged residue increase the resistance towards LAS.

As described above the type of mutation and the resistance towards LASfor variants with decreased LAS resistance is summarized in thefollowing table:

Relative Variant LAS resistance [%] Humicola insolens 100 Humicolainsolens/Y147H 71 Humicola insolens/E192P 52 Humicola insolens/D161P,A162P 64 Humicola insolens/D67T 44 Humicola insolens/Q36T, D67T 67Humicola insolens/D66N 47 Humicola insolens/D67N 71 Humicolainsolens/V64R 58 Humicola insolens/N65R 48 Humicola insolens/T93R 60Humicola insolens/Q36T, D67T, A83T 64 Humicola insolens/E91Q 71 Humicolainsolens/A191K 63 Humicola insolens/D42W 67 Humicola insolens/S117K 62Humicola insolens/R4H, A63R, N65R, D67R 54 Humicola insolens/D133N 0Humicola insolens/D58A 15 Humicola insolens/D67R 39 Humicolainsolens/A63R 38 Humicola insolens/R37N, D58A 6 Humicola insolens/K175R32 Humicola insolens/D2N 43 Humicola insolens/N65R, D67R 40 Humicolainsolens/T136D, G141R 5 Humicola insolens/Y147K 17 Humicolainsolens/Y147R 1 Humicola insolens/D161P 35 Humicola insolens/D66P 40Humicola insolens/D66A, D67T 39 Humicola insolens/D67T, *143NGT 7Humicola insolens/Q36T, D67T, *143NGT 0 Humicola insolens/N65R, D67R,S76K 22 Humicola insolens/W62R 25 Humicola insolens/S117R, F120S 31Humicola insolens/K13R 16 Humicola insolens/D10E 0

From this table it is seen that mutations of residues resulting in theintroduction of positively charged residue and/or the removal of anegatively charged residue decrease the resistance towards LAS.

Example 9 Alteration of pH Activity Profile

The pH activity profile of a cellulase is governed by the pH dependentbehavior of specific titratable groups, typically the acidic residues inthe active site. The pH profile can be altered by changing theelectrostatic environment of these residues, either by substitution ofresidues involving charged or potentially charged groups such as Arg(R), Lys (K), Tyr (Y), His (H), Glu (E), Asp (D) or Cys (C) if notinvolved in a disulphide bridge or by changes in the surfaceaccessibility of these specific titratable groups by mutations in thebiding cleft within 5 Å of the substrate.

In this example Humicola insolens cellulase and variants of Humicolainsolens cellulase involving substitution of charged or potentiallycharged residues have been tested for activity towards PASC at pH 7 andpH 10, respectively.

In order to determine the pH optimum for cellulases we have selectedorganic buffers because it is common known that, e.g., borate formscovalent complexes with mono- and oligo-saccharides and phosphate canprecipitate with Ca-ions. In DATA FOR BIOCHEMICAL RESEARCH Third EditionOXFORD SCIENCE PUBLICATIONS page 223 to 241, suitable organic buffershave been found. In respect of their pKa values we decided to useNa-acetate in the range 4-5.5, MES at 6.0, MOPS in the range 6.5-7.5,Na-barbiturate 8.0-8.5 and glycine in the range 9.0-10.5.

Method:

The method is enzymatic degradation of carboxy-methyl-cellulose, atdifferent pH's. Buffers are prepared in the range 4.0 to 10.5 withintervals of 0.5 pH unit. The analysis is based on formation of newreducing ends in carboxy-methyl-cellulose, these are visualized byreaction with PHBAH in strong alkaline environment, were they forms ayellow compound with absorption maximum at 410 nm.

Experimental Protocol;

Buffer preparation: 0.2 mol of each buffer substance is weighed out anddissolved in 1 liter of Milli Q water. 250 ml 0.2 M buffer solution and200 ml Milli Q water is mixed. The pH is measured using Radiometer PHM92labmeter calibrated using standard buffer solutions from Radiometer. ThepH of the buffers are adjusted to actual pH using 4 M NaOH or 4 M HCland adjusted to total 500 ml with water. When adjusting Na-barbiturateto pH 8.0 there might be some precipitation, this can be re-dissolved byheating to 50° C.

Acetic acid 100% 0.2 mol=12.01 g.MES 0.2 mol=39.04 g.MOPS 0.2 mol=41.86 g.Na-barbiturate 0.2 mol=41.24 g.Glycine 0.2 mol=15.01 g.

Buffers: pH: 4.0, 4.5, 5.0 & 5.5 Na-acetate 0.1 M pH: 6.0 Na-MES 0.1 MpH: 6.5, 7.0 & 7.5 Na-MOPS 0.1 M pH: 8.0 & 8.5 Na-barbiturate 0.1 M

pH: 9.0, 9.5, 10.0 & 10.5 Na.glycine 0.1 MThe actual pH is measured in a series treated as the main values, butwithout stop reagent, pH is measured after 20 min. incubation at 40° C.

Substrate Preparation:

2.0 g CMC, in 250 ml conic glass flask with a magnet rod, is moistenedwith 2.5 ml. 96% ethanol, 100 ml. Milli Q water is added and then boiledto transparency on a heating magnetic stirrer. Approximately 2 min.boiling. Cooled to room temperature on magnetic stirrer.

Stop Reagent;

1.5 g PHBAH and 5 g K-Na-tartrate dissolved in 2% NaOH.

Procedure:

There are made 3 main values and 2 blank value using 5 ml glass testtubes. (1 main value for pH determination)

Main values Blank value Buffer 1.0 ml. 1.0 ml. Substrate CMC 0.75 ml.0.75 ml. Mix 5 sec. 5 sec. Preheat 10 min./40° C. — Enzyme 0.25 ml. —Mix 5 sec. — Incubation 20 min./40° C. room temp. PHBAH-reagent 1 ml. 1ml. Mix 5 sec. — Enzyme — 0.25 ml. Mix — 5 sec.

Mixing on a Heidolph REAX 2000 mixer with permanent mix and maximumspeed (9). No stirring during incubation on water bath with temperaturecontrol. Immediately after adding PHBAH-reagent and mixing the samplesare boiled 10 min. Cooled in cold tap water for 5 min, Absorbance readat 410 nm.

Determination of Activity

The absorbance at 410 nm from the 2 Main values are added and divided by2 and the 2 Blank values are added and divided by 2, the 2 mean valuesare subtracted. The percentages are calculated by using the highestvalue as 100%.

The measured pH is plotted against the relative activity.

Buffer Reagents:

Acetic acid 100% from MERCK cat.no.1.00063, batch no. K20928263 422, pKa4.76, MW 60.05;MES (2[N-Morpholino]ethanesulfonic acid) from SIGMA cat.no. M-8250,batch no. 68F-5625, pKa 6.09: MW 195.2;MOPS (3-[N-Morpholino]propanesulfonic acid) from SIGMA cat.no. M-1254,batch no. 115F-5629, pKa 7.15, MW 209.3;Na-barbiturate (5,5-Diethylbarbituric acid sodium salt) from MERCKcat.no. 6318, batch no. K20238018 404, pKa 7.98, MW 206.2;Glycine from MERCK cat.no.4201, batch no. K205535601 405, pKa 9.78, MW75.07; PHBAH (p-hydroxy benzoic acid hydrazide) from SIGMAcat.no.H-9882, batch no. 53H7704;

-   K-Na-tartrate (Potassium sodium tartrate tetrahydrate) from MERCK    cat.no. 8087, batch no. A653387 304;    NaOH (Sodium hydroxyde) from MERCK cat.no. 1.06498, batch no.    C294798 404;    CMC (Carboxy Methyl Cellulose) supplied by Hercules (FMC)7LF    (November 1989).

Cellulase resistance to anionic surfactants was measured as activity onPASC (phosphoric acid swollen cellulose) at neutral pH (pH 7.0) vs.activity on PASC at alkaline pH (pH 10.0).

The reaction medium contained 5.0 g/l of a commercial regular powderdetergent from the detergent manufacturer NOPA Denmark. The pH wasadjusted to pH 7.0 and pH 10.0, respectively. Further the reactionmedium included 0.5 g/l PASC, and the reaction proceeded at thetemperature 30° C. for 30 minutes. Cellulase was dosed at 0.20 S-CEVU/I.After the 30 minutes of incubation the reaction was stopped with 2 NNaOH and the amount of reducing sugar ends determined through reductionof p-hydroxybenzoic acid hydrazide. The decrease in absorption ofreduced p-hydroxybenzoic acid hydrazide relates to the cellulaseactivity.

Results:

The results are presented in the table below, the activity at pH 10relative to pH 7 is compared to that of wild type Humicola insolenscellulase.

PASC activity pH 10/pH 7 Variant relative to wild type [%] Humicolainsolens 100 Humicola insolens/S76K, S117D 120 Humicola insolens/V129L133 Humicola insolens/R4H, A63R, N65R, D67R 120 Humicola insolens/R252L,Y280F 115 Humicola insolens/D161P, A162P 117 Humicola insolens/A57C,A162C 110 Humicola insolens/S76K 117 Humicola insolens/D161P, A162P,R196E 113 Humicola insolens/Q36T, D67T, A83T 111 Humicola insolens/W62R112 Humicola insolens/D42Y 110 Humicola insolens/S76K, A78K 114 Humicolainsolens/S76K, A78R 118

From the above table it is seen that the relative alkaline activity canbe increased by creating variants involving potentially charged residuesand/or by altering residues in the binding cleft less that 5 Å from thesubstrate.

Similarly the following table shows that the relative acidic activitycan be increased by other mutations involving potentially chargedresidues and/or by altering residues in the binding cleft less than 5 Åfrom the substrate.

PASC activity pH 10/pH 7 relative Variant to wild type [%] Humicolainsolens 100 Humicola insolens/D58A 83 Humicola insolens/Y280W 90Humicola insolens/D67R 89 Humicola insolens/A63R 85 Humicolainsolens/Y8F 82 Humicola insolens/W62E 82 Humicola insolens/R37N, D58A84 Humicola insolens/K175G 81 Humicola insolens/K175R 82 Humicolainsolens/Y8F, M104Q 83 Humicola insolens/Y8F, W62E, R252L, Y280F 83Humicola insolens/W62E, A162P 87 Humicola insolens/Y8F, W62E, A162P, 88Humicola insolens/Y147H 90 Humicola insolens/Y147N 90 Humicolainsolens/Y147Q 85 Humicola insolens/Y147W 85 Humicola insolens/E192P 89Humicola insolens/R158G 83 Humicola insolens/S152D 90 Humicolainsolens/K13Q 82 Humicola insolens/R37P 82 Humicola insolens/S45T 87Humicola insolens/E48D 86 Humicola insolens/R7I 83 Humicolainsolens/P14A 84 Humicola insolens/A19G 90 Humicola insolens/A19T 90Humicola insolens/R4H, D161P, A162P, R196E 88 Humicola insolens/D133N 80Humicola insolens/D40N 40 Humicola insolens/Y90F 72 Humicolainsolens/A63D 78 Humicola insolens/G127S, 25 I131A, A162P, Y280F, R252LHumicola insolens/Y147S 39 Humicola insolens/Y147F 71 Humicolainsolens/T6S 44 Humicola insolens/S55E 14 Humicola insolens/N123D 35Humicola insolens/N123Y 71 Humicola insolens/R158E 78 Humicolainsolens/T136D, G141R 57 Humicola insolens/G127S, I131A, A162P 52Humicola insolens/W62E, 35 G127S, I131A, A162P, Y280F, R252L Humicolainsolens/W62E, G127S, I131A, A162P 58 Humicola insolens/W62E, G127S,I131A 64 Humicola insolens/W62E, G127S, 80 I131A, Y280F, R252L Humicolainsolens/H119Q 57 Humicola insolens/Y8F, W62E 61 Humicola insolens/W62E,A162P 76 Humicola insolens/W62E, A162P 80 Humicola insolens/R158E, A162P80 Humicola insolens/Y8F, Y147S 63 Humicola insolens/Y147R 54 Humicolainsolens/Y147V 22 Humicola insolens/Y147C 67 Humicola insolens/Y147D 60Humicola insolens/N154D 74 Humicola insolens/R158E, R196E 79 Humicolainsolens/R158E, D161P, A162P 70 Humicola insolens/D67T, *143NGT 65Humicola insolens/Q36T, D67T, *143NGT 53 Humicola insolens/143*NGW,Q145D 53 Humicola insolens/L142P, 143*NGW, Q145E 42 Humicolainsolens/N65R, D67R, S76K 60 Humicola insolens/A63R, N65R, D67R 77Humicola insolens/T93R 80 Humicola insolens/S76R 70 Humicolainsolens/S117R, F120S 58 Humicola insolens/N123Q 63 Humicolainsolens/N123M 49 Humicola insolens/N123A 80 Humicola insolens/E48D,P49* 66 Humicola insolens/S55Y 61 Humicola insolens/S55M 48 Humicolainsolens/W18F 54 Humicola insolens/S45N 71 Humicola insolens/R7W 58Humicola insolens/K13R 72 Humicola insolens/R7L 74 Humicolainsolens/S15T 38 Humicola insolens/W18Y 37 Humicola insolens/C16M, C86G67 Humicola insolens/K13L 59 Humicola insolens/C12M, C47G 12 Humicolainsolens/W9F 62 Humicola insolens/C47G 58 Humicola insolens/C12G, C47M 0Humicola insolens/D10E 0 Humicola insolens/R7K 49

Accordingly, this example demonstrates that the relative activity pHprofile can be altered towards acidic or alkaline pH by creation ofvariants involving potentially charged residues and/or by alteringresidues in the binding cleft less that 5 Å from the substrate.

Example 10

Wash Performance of Cellulases Made Resistant to Anionic Surfactants

Application effect of a cellulase made resistant to anionic surfactantsvs. application effect of the native cellulase was measured as ‘colorclarification’ of worn black cotton swatches laundered with cellulase ina 0.1 liter mini-Terg-o-Meter. Laundering was done in varyingconcentrations of anionic surfactant.

The reaction medium contained phosphate buffer pH 7.0 and varyingconcentrations of LAS in the range 0.2-1.0 g/L. Two swatches werelaundered at 40° C. for 30 minutes, rinsed and then dried. Thislaundering cycle was repeated four times. All enzymes were tested ateach LAS concentration.

Finally the black cotton swatches were graded against a standard ofsimilar swatches washed with varying dosages of the native cellulase,the fungal ˜43 kD endo-beta-1,4-glucanase from Humicola insolens, DSM1800, (commercially available under the tradename Carezyme®), and theeffect expressed in PSU (panel score units).

LAS concentration 0.2 Variant g/l 0.4 g/l 0.6 g/l 0.8 g/l 1.0 g/lHumicola insolens 15 0 0 0 0 Humicola insolens/R158E 30 14 30 22 11Humicola insolens/R158G 20 18 20 33 28

1-78. (canceled)
 79. A method of producing an enzyme variant, comprisingintroduction a mutation in the amino acid sequence of a parentendoglucanase at one or more positions selected from the groupconsisting of: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 21a, 22, 24, 25, 26, 28, 29, 32, 33, 34, 35, 37, 40, 42,42a, 43, 44, 45, 47, 48, 49, 49a, 49b, 50, 53, 54, 55, 58, 59, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 79, 80, 82, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 95, 95d, 95h, 95j, 96, 97, 98, 99, 100,101, 102, 103, 104, 106, 110, 111, 112, 113, 114, 115, 116, 117, 119,121, 123, 127, 128, 129, 130, 131, 132, 132a, 133, 134, 136, 137, 138,139, 140, 140a, 141, 143a, 145, 146, 147, 148, 149, 150b, 150e, 150j,151, 152, 153, 154, 155, 156, 157, 158, 159, 160c, 160e, 160k, 161, 162,163, 164, 165, 166, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 181, 183, 184, 185, 186, 188, 191, 192, 193, 195, 196,197, 200, and 201, wherein each mutation is independently asubstitution, insertion or deletion, the enzyme variant hasendoglucanase activity and each position is numbered according to theamino acid sequence of the cellulase of SEQ ID NO:
 1. 80. The method ofclaim 79, wherein the parent endoglucanase comprises the amino acidsequence of SEQ ID NO:
 1. 81. The method of claim 79, wherein themutation is one or more of the following: K13L or L13K; P14A or A14P;S15H or H15S; K20E, K20G, K20A, E20K, G20K, A20K, E20G, E20A, G20E,A20E, G20A, or A20G; K21N or N21K; A22G, A22P, G22A, P22A, G22P, orP22G; V24*, V24L, *24V, L24V, *24L, or L24*; V28A, V28L, A28V, L28V,A28L, or L28A; N32D, N32S, N32K, D32N, S32N, K32N, D32S, D32K, S32D,K32D, S32K, or K32S; N34D or D34N; I38L, I38F, I38Q, L38I, F38I, Q38I,L38F, L38Q, F38L, Q38L, F38Q, or Q38F; S45N or N45S; G46S or S46G; E48D,E48N, D48E, N48E, D48N, or N48D; G50N or N50G; A53S, A53G, A53K, S53A,G53A, K53A, S53G, S53K, G53S, K53S, G53K, or K53G; Y54F or F54Y; W62F orF62W; A63D or D63A; V64I, V64D, I64V, D64V, I64D, or D64I; N65S, N65D,N65E, S65N, D65N, E65N, S65D, S65E, D65S, E65S, D65E, or E65D; D66N,D66P, D66T, N66D, P66D, T66D, N66P, N66T, P66N, T66N, P66T, or T66P;F68V, F68L, F68T, F68P, V68F, L68F, T68F, P68F, V68L, V68T, V68P, L68V,T68V, P68V, L68T, L68P, T68L, P68L, T68P, or P68T; A69S, A69T, S69A,T69A, S69T, or T69S; L70Y or Y70L; G71A or A71G; F72W, F72Y, W72F, Y72F,W72Y, or Y72W; A73G or G73A; A74F or F74A; T75V, T75A, T75G, V75T, A75T,G75T, V75A, V75G, A75V, G75V, A75G, or G75A; G79T or T79G; W85T or T85W;A88Q, A88G, A88R, Q88A, G88A, R88A, Q88G, Q88R, G88Q, R88Q, G88R, orR88G; Y90F or F90Y; L92A or A92L; T93Q, T93E, Q93T, E93T, Q93E, or E93Q;T95E or E95T; S96T or T96S; G97T, G97A, T97G, A97G, T97A, or A97T; P98Aor A98P; V99L or L99V; M104L or L104M; V106F or F106V; S110N or N110S,T111I, T111V, I111T, V111T, I111V, or V111I; G113Y or Y113G; L115V orV115L; G116S, G116Q, S116G, Q116G, S116Q, or Q116S; N118T, N118G, N118Q,T118N, G118N, Q118N, T118G, T118Q, G118T, Q118T, G118Q, or Q118G; H119Q,H119N, Q119H, or N119H; V129L or L129V; I131L, I131A, L131I, A131I,L131A, or A131L; G134A or A134G; Q138E or E138Q; G140N or N140G; R146Qor Q146R; S152D or D152S; R153K, R153L, R153A, K153R, L153R, A153R,K153L, K153A, L153K, A153K, L153A, or A153L; L163V, L163W, V163L, W163L,V163W, or W163V; G166S or S166G; W169F or F169W; R170F or F170R; F171Y,F171A, Y171F, A171F Y171A, or A171Y; D172E, D172S, E172D, S172D, E172S,or S172E; W173E or E173W; F174M, F174W, M174F, W174F, M174W, or W174M;A177N or N177A; D178P or P178D; N179V or V179N; P180L or L180P; L1931 orI193L; R1961, R196K, I196R, K196R, I196K, or K196I, and/or T197S orS197T.
 82. The method of claim 79, wherein the mutation is one or moreof the following: in position 4 holds R, H, K, Q, V, Y, or M; inposition 5 holds S, T, or A; in position 13 holds K, or L; in position14 holds P, or A; in position 15 holds H, or S; in position 16 holds C,or A; in position 19 holds A, D, S, P, T, or E; in position 20 holds A,E, G, or K; in position 21 holds K, or N; in position 21a holds V or *;in position 22 holds A, G, or P; in position 24 holds L, V, or *; inposition 28 holds A, L, or V; in position 32 holds D, K, N, or S; inposition 34 holds D or N; in position 38 holds F, I, L, or Q; inposition 42 holds D, G, T, N, S, K, or *; in position 44 holds K, V, R,Q, G, or P; in position 45 holds N, or S; in position 46 holds G, or S;in position 47 holds C, or Q; in position 48 holds D, E, N, or S; inposition 49 holds P, S, A, G, or *; in position 49a holds C, or *; inposition 49b holds N, or *; in position 50 holds G, or N; in position 53holds A, G, K, or S; in position 54 holds F, or Y; in position 62 holdsF, or W; in position 63 holds A, or D; in position 64 holds D, I, or V;in position 65 holds D, E, N, or S; in position 68 holds D, N, P, or T;in position 69 holds A, S, or T; in position 70 holds L, or Y; inposition 71 holds A, or G; in position 72 holds F, W, or Y; in position73 holds A, or G; in position 74 holds A, or F; in position 75 holds A,G, T, or V; in position 79 holds G, or T; in position 82 holds E, or *;in position 88 holds A, G, Q, or R; in position 90 holds F, or Y; inposition 92 holds A, or L; in position 93 holds E, Q, or T; in position95 holds E, or T; in position 95j holds P, or *; in position 96 holds S,or T; in position 97 holds A, G, or T; in position 98 holds A, or P; inposition 99 holds L, or V; in position 104 holds L, or M; in position106 holds F, or V; in position 110 holds N, or S; in position 111 holdsI, T, or V; in position 113 holds G, or Y; in position 115 holds L, orV; in position 116 holds G, Q, or S; in position 118 holds G, N, Q, orT; in position 119 holds H, N, or Q; in position 129 holds L, or V; inposition 131 holds A, I, or L; in position 132 holds A, P, or T; inposition 133 holds D, K, N, or Q; in position 134 holds A, or G; inposition 138 holds E, or Q; in position 145 holds A, D, N, or Q; inposition 146 holds Q, or R; in position 150b holds A, or *; in position152 holds D, or S; in position 153 holds A, K, L, or R; in position 163holds L, V, or W; in position 166 holds G, or S; in position 169 holdsF, or W; in position 170 holds F, or R; in position 171 holds A, F, orY; in position 172 holds D, E, or S; in position 173 holds E, or W; inposition 174 holds F, M, or W; in position 177 holds A, or N; inposition 178 holds D, or P; in position 179 holds N, or V; in position180 holds L, or P; in position 193 holds I, or L; in position 196 holdsI, K, or R; and/or in position 197 holds S, or T.
 83. The method ofclaim 79, wherein the mutation is one or more of the following;C16M+C86G, D42W, D42Y, E48D/P49*, E48N1/P49* and/or L70Y.
 84. The methodof claim 79, wherein the mutation is one or more of the following: K13Lor L13K; P14A or A14P; S15H or H15S; K20E, K20G, K20A, E20K, G20K, A20K,E20G, E20A, G20E, A20E, G20A, or A20G; K21N or N21K; A22G, A22P, G22A,P22A, G22P, or P22G; V24*, V24L, *24V, L24V, *24L, or L24*; N32D, N32S,N32K, D32N, S32N, K32N, D32S, D32K, S32D, K32D, S32K, or K32S; N34D orD34N; G50N or N50G; A53S, A53G, A53K, S53A, G53A, K53A, S53G, S53K,G53S, K53S, G53K, or K53G; Y54F or F54Y; V64I, V64D, I64V, D64V, I64D,or D64I; F68V, F68L, F68T, F68P, V68F, L68F, T68F, P68F, V68L, V68T,V68P, L68V, T68V, P68V, L68T, L68P, T68L, P68L, T68P, or P68T; A69S,A69T, S69A, T69A, S69T, or T69S; L70Y or Y70L; G71A or A71G; G79T orT79G; W85T or T85W; A88Q, A88G, A88R, Q88A, G88A, R88A, Q88G, Q88R,G88Q, R88Q, G88R, or R88G; L92A or A92L; T93Q, T93E, Q93T, E93T, Q93E,or E93Q; V106F or F106V; Q138E or E106Q; G140N or N140G; S152D or D152S;R153K, R153L, R153A, K153R; L153R, A153R, K153L, K153A, L153K, A153K,L153A, or A153L; G166S or S166G; W169F or F169W; R170F or F170R; F171Y,F171A, Y171F, A171F, Y171A, or A171Y; D172E, D172S, E172D, S172D, E172S,or S172E; W173E or E173W; F174M, F174W, M174F, W174F, M174W, or W174M;L193I or I193L; and/or T197S or S197T.
 85. The method of claim 79,wherein the mutation is one or more of the following: the amino acidresidue at position 22 is A, G, or P; the amino acid residue at position24 is L, V, or *; the amino acid residue at position 32 is D, K, N, orS; the amino acid residue at position 34 is D or N; the amino acidresidue at position 42 is D, G, T, N, S, K, or *; the amino acid residueat position 49 is P, S, A, G, or *; the amino acid residue at position49a is C or *; the amino acid residue at position 49b is N or *; theamino acid residue at position 50 is G or N; the amino acid residue atposition 53 is A, G, K, or S; the amino acid residue at position 54 is For Y; the amino acid residue at position 64 is D, I, or V; the aminoacid residue at position 68 is D, N, P, or T; the amino acid residue atposition 69 is A, S, or T; the amino acid residue at position 70 is L orY; the amino acid residue at position 71 is A or G; the amino acidresidue at position 79 is G or T; the amino acid residue at position 88is A, G, Q, or R; the amino acid residue at position 92 is A or L; theamino acid residue at position 93 is E, Q, or T; the amino acid residueat position 95j is P or *; the amino acid residue at position 106 is For V; the amino acid residue at position 138 is E or Q; the amino acidresidue at position 150b is A or *; the amino acid residue at position152 is D or S; the amino acid residue at position 153 is A, K, L, or R;the amino acid residue at position 166 is C or S; the amino acid residueat position 169 is F or W; the amino acid residue at position 170 is For R; the amino acid residue at position 171 is A, F, or Y; the aminoacid residue at position 172 is D, E, or S, the amino acid residue atposition 173 is E or W; the amino acid residue at position 174 is F, M,or W; the amino acid residue at position 193 is I or L; and/or the aminoacid residue at position 197 is S or T.